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WO2025179343A1 - Quantitative nucleic acid amplification and detection apparatus and methods thereof - Google Patents

Quantitative nucleic acid amplification and detection apparatus and methods thereof

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Publication number
WO2025179343A1
WO2025179343A1 PCT/AU2025/050174 AU2025050174W WO2025179343A1 WO 2025179343 A1 WO2025179343 A1 WO 2025179343A1 AU 2025050174 W AU2025050174 W AU 2025050174W WO 2025179343 A1 WO2025179343 A1 WO 2025179343A1
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WIPO (PCT)
Prior art keywords
temperature
amplification
test mixture
time
sample
Prior art date
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Pending
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PCT/AU2025/050174
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French (fr)
Inventor
William Robb Hopper
Jonathan Scott RICHARDS
Jack Maxwell HOPPER
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AXXIN Pty Ltd
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AXXIN Pty Ltd
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Publication date
Priority claimed from AU2024900483A external-priority patent/AU2024900483A0/en
Application filed by AXXIN Pty Ltd filed Critical AXXIN Pty Ltd
Publication of WO2025179343A1 publication Critical patent/WO2025179343A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present disclosure generally relates to the field of nucleic acid amplification and detection, such as nucleic acid amplification to detect and quantify the presence of one or more specific nucleic acids in a sample.
  • the present disclosure also relates to methods of quantitative isothermal nucleic acid amplification and detection apparatus.
  • the present disclosure further relates to quantitative nucleic acid amplification and detection apparatus and methods using temperature ramping and cycling.
  • nucleic acid detection and quantification is an important part of many biomedical, environmental, veterinary, forensic and food safety processes. In particular, nucleic acid detection has become increasingly important in the discovery of genetic diseases, diagnosing pathogenic infections and monitoring disease treatment.
  • nucleic acid molecules are typically present in low concentrations in biological samples, thus nucleic acid detection consists of an amplification step and a detection step.
  • the most common amplification technique is polymerase chain reaction (PCR), which is the gold- standard for detecting nucleic acids in samples due to its reliability and specificity.
  • PCR can amplify a single or a few copies of DNA by several orders of magnitude, generating thousands to millions of copies of the DNA sequence.
  • Two essential components of a PCR reaction are primers containing sequences complementary to the target region and DNA polymerase.
  • PCR relies on thermal cycling to proceed through the steps of denaturation of the double- stranded DNA (dsDNA), annealing of primers, and extension of the primer along the template by a thermostable polymerase.
  • dsDNA double- stranded DNA
  • primers primers
  • extension of the primer along the template by a thermostable polymerase.
  • the nucleic acid molecules double approximately each full temperature cycle, as each nucleic acid molecule is used as a template to form a new copy.
  • the overall quantity of nucleic acids produced over a number of cycles follows an exponential curve.
  • PCR methods use thermal cycling, or a series of heating and cooling steps and the use of a heat-stable DNA polymerase, such as Taq polymerase (originally isolated from Thermits aquatic us), which enzymatically assembles a new DNA strand using nucleotides, single-stranded DNA as a template and primers.
  • a heating step separates the two DNA strands process (DNA melting). At a lower temperature, each strand is used as a template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA.
  • the selectivity of PCR results from use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
  • Quantitative PCR (or also known as real-time PCR or quantitative realtime PCR) is a PCR-based technique that provides amplification and quantification of the nucleic acid species in a sample.
  • fluorescence is measured after each cycle and the intensity of the fluorescent signal reflects the momentary amount of nucleic acid in the sample at that specific time. In the initial cycles, the fluorescence signal is too low to be detected from the background signal. Once a sufficient number of amplification cycles have occurred to increase the fluorescence signal above the background signal a measurement of the portion of the initial amount of template nucleic acid in the sample can be determined.
  • thermocycler for temperature cycling and detection, which is both expensive and cumbersome in point- of-care testing settings. Additionally, there is a steep power requirement for running thermocycler units. Temperature jumps of up to 50°C may be required between successive steps of a PCR amplification reaction.
  • Isothermal amplification techniques have been developed for detection of nucleic acids. Isothermal amplification techniques are performed at a constant temperature thereby circumventing the need for temperature cycling. The optimal temperature for isothermal amplification varies depending on the reagents used and is commonly in the range of 30-65°C.
  • LAMP Loop mediated amplification
  • NEAR Nicking and Extension Amplification Reaction
  • TMA Transcription-Mediated Amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • SPIA Single Primer Isothermal Amplification
  • RCA Rolling Circle Amplification
  • HDA helicasedependent amplification
  • MDA multiple displacement amplification
  • SDA Strand displacement amplification
  • RPA recombinase polymerase amplification
  • Quantitative detection has been applied to isothermal processes, either by conventional real-time monitoring or product accumulation or by digital end point analysis of compartmentalized parallel reactions.
  • these methods have been unreliable for two reasons: (1) it is difficult to determine the starting point of the reaction as the reaction range is not taken into consideration and (2) there is variability in the conditions which results in the optimum temperature not being the same from day-to-day and from instrument-to-instrument.
  • the inventors sought to produce a method of accurately and reliably amplifying, detecting and/or quantifying nucleic acids in a sample during nucleic acid amplification.
  • the inventors found that when subjecting a sample to substantially isothermal conditions, they were able to accurately and reliably identify the amplification start time of the reaction by using a detection regent (e.g., a decaying temperature dependent fluorescent dye) or a threshold temperature.
  • a detection regent e.g., a decaying temperature dependent fluorescent dye
  • a threshold temperature e.g., a threshold temperature
  • the inventors also found that by performing ramping at a reduced ramping rate to standard thermocycling following the amplification start time, they were able to increase the time period between the amplification start time and detectable amplification curve, further increasing the accuracy of detection and quantification of nucleic acids in the sample.
  • the findings by the inventors provide the basis for methods of identifying an amplification start time of, amplifying, detecting and/or quantifying one or more nucleic acids in a sample.
  • Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • Some embodiments relate to a method for determining an initial quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • the at least one detection reagent may be at least one first fluorophore, wherein fluorescence emitted by the first fluorophore of the at least one fluorophore varies with nucleic acid binding, and wherein the detection reagent data comprises fluorescence emitted by the at least one first fluorophore of the test mixture.
  • the at least one fluorophore may be at least one decaying temperature dependent fluorescent dye.
  • the decaying temperature dependent fluorescent dye is Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
  • subjecting the test mixture to substantially isothermal conditions comprises heating the test mixture to a temperature of between 20 and 65 degrees Celsius.
  • the temperature may be at about 40 degrees Celsius or at about 65 degrees Celsius.
  • the substantially isothermal condition is at a temperature of about 40 degrees Celsius, and during ramping the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius;
  • the substantially isothermal condition is at a temperature of about 65 degrees Celsius, and during ramping the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
  • a temperature difference between the first temperature and the second temperature is between 2 and 40 degrees Celsius.
  • the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius;
  • the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
  • the temperature may be ramped at a ramping rate of between 0.01 degrees Celsius per second and 5 degrees Celsius per second. During ramping the first temperature may be ramped for a period of approximately 100 seconds and/or the second temperature may be ramped for a period of approximately 90 seconds.
  • recording, over the first time period, data indicative of the temperature of the test mixture comprises receiving temperature measurements from a sensor of the apparatus configured to sense, directly or indirectly, a temperature of the test mixture.
  • the start time detection condition may be a threshold range of the amplification temperature.
  • the test mixture comprises at least one at least one second fluorophore that varies with temperature, and recording, over the first time period, data indicative of the temperature of the test mixture comprises determining fluorescence emitted by the at least one second fluorophore over the first time period.
  • the at least one second fluorophore may be the at least one first fluorophore.
  • the start time detection condition may be a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature.
  • the start time detection condition may be a maximum rate of change of gradient of the at least one second fluorophore.
  • the start time detection condition may be a steady state emission level of fluorescence, the steady state emission level being indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids.
  • the method further comprises calculating the quantity of the at least one nucleic acid based on the respective amplification duration.
  • the amplification duration may be the time difference between the detection point and the start time.
  • the amplification duration may be a number of ramping cycles.
  • the method further comprises comparing the amplification time duration to standards indicative of known amplification time durations for known concentrations of nucleic acids to calculate the quantity of the at least one nucleic acid.
  • the test mixture may further comprise a DNA polymerase with stranddisplacement activity.
  • the DNA polymerase is selected from the group consisting of phi29 or Bsu large fragment and Bst.
  • the test mixture may comprise an additive to lower the melting temperature (Tm) of the one or more nucleic acids in the sample, the additive may be betaine, for example.
  • Tm melting temperature
  • the one or more nucleic acids may be DNA or RNA.
  • the one or more nucleic acids may be RNA and the test mixture may further comprise a reverse transcriptase.
  • Some embodiments relate to a method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture, and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • Some embodiments relate to a method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
  • Some embodiments relate to a system comprising one or more processors; and memory comprising computer executable instructions, which when executed by the one or more processors, cause the system to perform any one or more of the described methods.
  • Some embodiments relate to a computer-readable storage medium storing instructions that, when executed by a computer, cause the computer to perform any one or more of the described methods.
  • the computer-readable storage medium may be a nontransient computer-readable storage medium.
  • Figure 1 is a series of graphical representations showing isothermal amplification measurements as a function of temperature.
  • A shows the rate at which isothermal amplification occurs for different reaction temperatures and
  • B shows isothermal amplification curves produced at reaction temperatures marked, where for each temperature (tempA, tempB, tempC, tempO), the same four initial target DNA concentrations are amplified.
  • Figure 2 is a graphical representation showing isothermal amplification when the block/amplification liquid temperature is set at a non-optimal temperature for amplification.
  • Figure 3 is a graphical representation showing an exemplary method of the present disclosure.
  • (A) shows the block temperature increases (ramps up) and decreases (ramps down) between an upper temperature (tempB) and a lower temperature (tempA) (shown as solid lines) and (B) show the temperature of the reaction as a result of the ramping in block temperature cycles through the optimal temperature range. When enough cycles or ramps have occurred, sufficient amplification will allow for detection of nucleic acids in the sample (detection point).
  • Figure 4 is a graphical representation showing amplification fluorescence as the reaction proceeds through multiple ramps.
  • the concentration of nucleic acids within the sample slowly increases through repeated cycles of ramps and reaches a point at which the fluorescence can be detected.
  • Figure 5 is a series of graphical representations showing an exemplary method of quantification in isothermal amplification. The method involves interpolating between
  • FIG. 6 is a block diagram showing a nucleic acid amplification and detection apparatus, according to some embodiments.
  • Figures 7A to D show an exemplary nucleic acid amplification and detection apparatus of the present disclosure.
  • Figure 7A show a perspective view of the apparatus with a sample receiving panel in a closed state
  • Figure 7B shows a perspective view of the apparatus with the sample receiving panel in an open state and showing multiple samples received by the apparatus
  • Figure 7C shows the apparatus with the sample receiving panel in the open state and showing a single sample received by the apparatus
  • Figure 7D shows the apparatus with the sample receiving panel in the open state and showing a single sample in a closed cap tube received by the apparatus.
  • Figures 8A and 8B is an exploded view of an exemplary nucleic acid amplification and detection apparatus including a circuit board and with the circuit board removed, respectively.
  • Figure 9 is an expanded view of the heating and cooling element of the nucleic acid amplification and detection apparatus.
  • Figure 10 is a graphical representation showing isothermal quantification with dilutions at 10 1 to 10’ 6 .
  • Fluorophore is HEX, on T8 Axxin machine with beads, optimum temperature 65 degrees Celsius. Circled region shows starting region of the curve.
  • Figure 11 is a graphical representation showing determination of amplification start time by interpreting the fluorescence warm up curve shape.
  • A Crossing point where the assay temperature crosses a defined level of fluorescence difference corresponding to temperature difference as the assay approaches the running temperature is shown by arrow A; Line at arrow B shows fixed level of fluorescence.
  • B Inflection point or point of maximum rate of change of gradient in fluorescence temperate response is shown at arrow C.
  • Figure 12 is a graphical representation of an isothermal assay with ramp cycling. Individual amplification time points of nucleic acids at different concentrations are shown.
  • Figure 13 is a graphical representation showing isothermal amplification with temperature ramping. Temperature record over time using isothermal amplification at 50 degrees Celsius for 15 mins followed by temperature cycling of 100 second ramp down to 50 degrees Celsius and 90 seconds at 65 degrees Celsius.
  • Figure 14 is a block diagram of a system for quantifying nucleic acid in a sample, according to some embodiments.
  • Figure 15 is a process flow diagram of a method of quantifying one or more nucleic acids in a sample, according to one embodiment.
  • Figure 16 is a process flow diagram of a method of determining an amplification process start time, according to a further embodiment.
  • Figure 17 is a process flow diagram of a method of determining one or more detection points indicative of sufficient amplification to allow for detection of one or more respective nucleic acids in the sample, according to a yet further embodiment.
  • Figure 18 is a process flow diagram of a method of quantifying one or more nucleic acids in the sample, according to a further embodiment.
  • reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.
  • reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.
  • sample collection and preparation and related technical techniques utilised in the present disclosure are standard procedures, well known to those skilled in the art.
  • nucleic acid has the meaning generally understood in the art and refers to DNA or RNA or both, including any modifications.
  • nucleotide sequence or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone.
  • amplification has the meaning as well-known in the art and refers to the process of making multiple copies of a nucleic acid or nucleic acid sequence.
  • amplification temperature refers to the temperature at which an amplification enzyme initiates, or is able to initiate, amplification.
  • the amplification temperature is a continuous variable and refers to the range of temperatures at which the amplification enzyme is active.
  • the “optimal amplification temperature” or “optimal temperature” refers to a temperature or small temperature range at which an amplification enzyme initiates an amplification reaction at an optimum (usually fastest) rate of reaction or rate of amplification.
  • the amplification temperature is optimised for the activity of the polymerase enzyme used in the reaction. It ensures efficient amplification of the target DNA or RNA sequence. The exact temperature depends on the specific isothermal method being used and the enzyme involved.
  • LAMP Loop-mediated isothermal amplification
  • RPA Recombinase Polymerase Amplification
  • NASBA Nucleic Acid Sequence Based Amplification
  • amplification time refers to the period of time required to amplify nucleic acids (DNA or RNA sequence) to a detectable level. The exact time depends on the specific isothermal method being used and the enzyme involved e.g., the activity of the polymerase enzyme used in the reaction.
  • period of time shall be understood to refer to an extent of time of any length. It will be apparent to the skilled person from the disclosure herein that the first and second periods of time need not necessarily be the same and may be different.
  • isothermal conditions in respect of a method of nucleic acid amplification refers to the continuous, exponential amplification of nucleic acids at a constant temperature.
  • the term “substantially isothermal conditions” refers to nucleic acid amplification at a single temperature or within a narrow range of temperatures that does not vary significantly.
  • the method is carried out at a temperature that varies by only about 1-5 degrees Celsius (e.g., varying by 1, 2, 3, 4, or 5 degrees Celsius).
  • the present disclosure provides a method of quantifying one or more nucleic acids in a sample forming part of a test mixture.
  • the present disclosure provides a method of quantifying one or more nucleic acids in a sample forming part of a test mixture, comprising subjecting the sample to substantially isothermal conditions.
  • the method comprises subjecting the sample to substantially isothermal conditions for a first period of time and/or a second period of time. In one example, the method comprises subjecting the sample to substantially isothermal conditions for a first period of time. In another example, the method comprises subjecting the sample to substantially isothermal conditions for a second period of time. In a further example, the method comprises subjecting the sample to substantially isothermal conditions for a first period of time and a second period of time.
  • isothermal amplification comprises continuous, exponential amplification of nucleic acids at a constant temperature using enzymes, for example but not limited to strand displacing polymerases, restriction enzymes and helicases, rather than temperature changes. Isothermal amplification techniques suitable for use in the present disclosure will be apparent to the skilled person and/or described herein.
  • LAMP Loop- mediated Amplification
  • NEAR Nicking and Extension Amplification Reaction
  • SDA Strand Displacement Amplification
  • RCA Rolling Circle Amplification
  • TMA Transcription-Mediated Amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • SPIA Single Primer Isothermal Amplification
  • HDA Helicasedependent Amplification
  • MDA Multiple Displacement Amplification
  • RPA Recombinase Polymerase Amplification
  • Loop-mediated Amplification is a common isothermal amplification technique that is both sensitive and specific.
  • LAMP employs a thermostable polymerase with strand displacement capabilities and four or more primers.
  • the primers are designed to anneal consecutively along the target in the forward and reverse direction. Extension of the outer primers displaces the extended inner primers to release single strands.
  • Each primer is designed to have hairpin ends that, once displaced, snap into a hairpin to facilitate self-priming and further polymerase extension.
  • LAMP is a very fast approach to synthesising a lot of DNA, in a very short period of time. Additional loop primers can decrease the amplification time but complicates the reaction mixture. LAMP is used commercially, for example, in rapid molecular tests and microorganism detection.
  • NEAR Nicking and Extension Amplification Reaction
  • a strand displacing polymerase a strand displacing polymerase
  • a restriction endonuclease a restriction endonuclease
  • One set of primers serve to displace the initially extended second set of primers to create a single- strand for the next primer to bind.
  • a restriction site is present in the 5' region of the primer.
  • enzymes also known as nicking endonucleases (NEases) which are enzymes derived from a mutation of restriction enzymes, and DNA polymerase.
  • DNA polymerases with strand displacement activity include Phi 29, Klenow Fragment, vent, and Bst DNA polymerase. Among these enzymes, the most commonly used is Bst DNA polymerase.
  • nicking enzyme-combined amplification includes the following four steps: (1) the nicking enzyme recognises and cleaves a specific site on a single strand in the double strand, exposing the 3' end; (2) the polymerase extends a new strand from the 3' end, the new strand containing the recognition site for the nicking enzyme; (3) the nascent strands may be used directly as a product but are more often used to participate in downstream reactions; (4) a large amount of target DNA/RNA product is obtained (Cao et al 2022).
  • NEAR can be divided into categories based on the combination of different types of isothermal amplification with nicking enzymes, for example, typical Nicking Enzyme-Combined Amplification (EXPAR), Nicking Enzymes Combined with SDA, Nicking Enzymes Combined with RCA, and NEAR on its own.
  • EXPAR typical Nicking Enzyme-Combined Amplification
  • SDA SDA
  • RCA Nicking Enzymes Combined with RCA
  • NEAR on its own.
  • Strand Displacement Amplification is a continuous nicking and polymerisation/displacement process catalysed by nicking enzymes, for example, restriction enzyme Hindi, and DNA polymerases, for example, an exonuclease-deficient DNA polymerase, Hexachloro-fluorescein (HEX) or Rhodamine B (RhB).
  • HEX Hexachloro-fluorescein
  • RhB Rhodamine B
  • the restriction endonuclease will nick the target DNA, allowing DNA polymerase to extend the 3’ end.
  • primer 1 primer 2
  • template 1 and template 2 two primertemplate complexes are formed after denaturation. Both primers contain an identification sequence that is used to cause the identification cleavage of the nicking enzyme.
  • DNA polymerase After being cut, DNA polymerase extends the 3' end of the double strand to generate dsDNA containing the complete nicking site, which will be cut by a nicking enzyme to generate a new 3' end at the notch, triggering a new extension reaction and causing the displacement of the downstream target strand.
  • the ssDNA from the primer 1 -template 1 complex can be used as a template for primer 2, and the product from the primer2- template2 complex can also be used as template for primer 1. This cycle results in exponential amplification of the target.
  • This technique can have some limitations compared to other isothermal techniques, including lower primer specificity.
  • Rolling Circle Amplification is an isothermal nucleic acid amplification method based on ligase binding, primer extension, and strand displacement amplification reaction [73], which mimics the rolling loop replication process of microbial circular DNA in nature. Under constant temperature conditions, many repetitive sequences complementary to the ring probe can be generated. After the isothermal linear amplification of the ring probe in vitro is realized in combination with the nicking enzyme, RCA can change from linear amplification to exponential amplification or multi-primer amplification. Only a segment of a nicking enzyme needs to be designed on the ring probe. With amplification, a long strand of DNA with a specific sequence interval is obtained. RCA is a linear amplification, but when nicking enzymes are used in conjunction with multiple templates, the amplification becomes exponential.
  • TMA Transcription-Mediated Amplification
  • RNA polymerase reverse transcription and subsequent generation of numerous transcripts by RNA polymerase. Following amplification, these RNA copies are hybridised with a complementary oligonucleotide probe for detection via a chemiluminescent tag.
  • TMA produces 100-1000 copies per cycle, resulting in a 10 billionfold increase within 15-30 min.
  • TMA is popular in clinical diagnostics with numerous commercial tests based on the technique available to clinical laboratories, and is also used in food and water safety, among other areas.
  • Nucleic Acid Sequence-Based Amplification is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature, for example at 41 degrees Celsius.
  • NASBA is a two-step process used to produce multiple copies of single stranded RNA by annealing specially designed primers, then utilising an enzyme cocktail to amplify it.
  • NASBA is also commonly referred to as “self- sustained sequence replication” whereby RNA is converted into cDNA, which is transcribed to produce more RNA.
  • NASBA routinely produces a high level of amplification, on the order to 10 9 .
  • NASBA has been used to develop rapid diagnostic tests for several pathogenic viruses with single- stranded RNA genomes, for example, influenza A, zika virus, foot-and-mouth disease virus, severe acute respiratory syndrome (SARS)-associated coronavirus, human bocavirus (HBoV) and also parasites like Trypanosoma brucei.
  • viruses with single- stranded RNA genomes, for example, influenza A, zika virus, foot-and-mouth disease virus, severe acute respiratory syndrome (SARS)-associated coronavirus, human bocavirus (HBoV) and also parasites like Trypanosoma brucei.
  • viruses with single- stranded RNA genomes for example, influenza A, zika virus, foot-and-mouth disease virus, severe acute respiratory syndrome (SARS)-associated coronavirus, human bocavirus (HBoV) and also parasites like Trypanosoma brucei.
  • SARS severe acute respiratory syndrome
  • HBV human bocavirus
  • Single Primer Isothermal Amplification is an approach using only one DNA-RNA chimeric primer along with RNase H and a DNA polymerase with strand displacement activity. This approach is capable of amplifying more DNA than PCR when primer concentrations are increased. Applications using this technique include on-site diagnosis and DNA detection.
  • MDA Multiple Displacement Amplification
  • HD A Helicase-dependent Amplification
  • MutL single- stranded DNA-binding protein
  • SSB single- stranded DNA-binding protein
  • Recombinase Polymerase Amplification amplifies DNA at a constant temperature (37-42C) using a recombinase, primers, a single-stranded DNA binding protein (SSB), and a strand displacing DNA polymerase.
  • the recombinase is complexed with the primer.
  • the complex is able to bind with doublestranded DNA at homologous sequences through a strand exchange.
  • a single-stranded binding protein, T4 gp32 stabilises the displaced strand.
  • Bsu polymerase extends the primers, creating a new complete copy of the template.
  • primers used in a method of the present disclosure will depend on the assay format used. Methods for designing primers for, for example, isothermal amplification methods disclosed herein are known in the art and described, for example, in Dieffenbach 1995.
  • a primer (or the sequence thereof) is assessed to determine the temperature at which it denatures from a target nucleic acid (i.e., the melting temperature of the probe or primer, or T m ).
  • the T m is defined as the temperature at which half of the DNA strands are in the random coil or single- stranded (ssDNA) state.
  • T m depends on the length of the DNA molecule and its specific nucleotide sequence. Methods of determining T m are known in the art and described, for example, in Santa Lucia, 1995 or Bresslauer et al., 1986.
  • the sample comprises an additive to lower the T m of the one or more nucleic acids in the sample.
  • additives suitable for lowering T m of nucleic acids include, for example, betaine and formamide.
  • the additive is betaine.
  • DNA polymerases are used to separate duplex DNA.
  • the rate at which isothermal amplification occurs varies depending on the temperature of the reaction fluid in which the polymerase enzyme is present.
  • the optimal temperature at which amplification will occur is dependent on the reagent constituents including the specific primers and polymerases used. The variation is partially due to the instrument-to-instrument variation as well as the ambient temperature in which the reaction sample is prepared. Additionally, even if the instrument was to run the sample at the exact same temperature, the next batch of reagents may have a peak that has a different optimal temperature range. Amplification will also occur for a range of temperatures above and below this optimal temperature but at slower rates.
  • the sample comprises a DNA polymerase with stranddisplacement activity.
  • the DNA polymerase is selected from the group consisting of phi29, Bsu large fragment and Bst.
  • DNA polymerases phi29 or Bsu large fragment may be used in moderate temperature reactions (20-40°C) or a Bst DNA polymerase may be used for higher temperature reactions (50-65°C). Primer-based annealing and extending are then performed.
  • the optimal temperature is the temperature at which nucleic acid amplification occurs most rapidly. Amplification will occur within a temperature range above and below this optimal temperature. This temperature range may only be a couple of degrees (e.g., 2-5 degrees Celsius) or could be as much as degrees, for example, between 60-70 degrees Celsius. Small changes in the amplification liquid temperature may result in a large range in the rate at which isothermal amplification will occur. The most rapid amplification occurs where the temperature of the amplification fluid is at the optimal temperature. Significant changes in amplification will affect the accuracy of the reference curve-based quantification.
  • the optimal temperature of the reaction is 55 degrees Celsius and during the sample run the instrument is running at 58 degrees Celsius, the reaction is not running at the optimal temperature, and this will result in a different quantification when compared to standards (which are generally not run on the same day). It will be apparent to the skilled person that running standards at the sub-optimal temperature is expensive and requires increased time for testing.
  • the non-optimal amplification fluid temperature setting method has a number of challenges. Firstly, it relies on precise knowledge of the temperature range at which isothermal amplification will occur for a specific amplification configuration. For example, when a non-optimal temperature is selected which is close to the edge of this range, and the range is not accurately determined, there is chance that false negatives will result. Secondly, this method may require accurate amplification fluid temperature control. For example, if a non-optimal temperature is selected for performing the quantified isothermal amplification, a relatively minor error in amplification fluid temperature may lead to large variations in the rate at which isothermal amplification will occur.
  • the method comprises identifying the amplification start time when a first detection reagent is at a threshold level.
  • a “detection reagent” is a substance or solution that reacts with certain other substances or substances in a characteristic manner. Typical characteristics include colour change, fluorescence, formation of a precipitation etc and suitable detection reagents are well-known in the art and/or described herein.
  • the first detection reagent is a temperature dependent fluorescent dye.
  • the first detection reagent is a decaying temperature dependent fluorescent dye.
  • temperature dependent fluorescent dyes produce fluorescent signals that either decay or increase in response to increasing temperature.
  • a decaying temperature dependent fluorescent dye is used, the fluorescent signal decreases as the temperature ramps up.
  • An exemplary temperature dependent dye is Rhodamine B (RhB) that when stimulated by an excitation source of between approximately 500nm-550nm, RhB produces a fluorescent signal with an emission peak in the range of 565nm to 590nm, which diminishes as temperature increases.
  • the start time detection condition is:
  • determining the start time comprises determining the time corresponding to the maximum rate of change of gradient of the second set of fluorescence data
  • the start time detection condition is a maximum rate of change of gradient of the fluorescent signal of a fluorophore, e.g., the decaying temperature dependent fluorescent dye.
  • the maximum rate of change of gradient of the fluorescent signal of the decaying temperature dependent fluorescent dye is determined by using a first derivative or a second derivative analysis.
  • the first derivative or second derivative is determined.
  • first derivative will be understood to refer to the rate of change of a functions output with respect to its input, such as the rate of change of fluorescence over time (i.e., dy/dx).
  • first derivative is the slope of the tangent to the fluorescent signal at each point.
  • the term “second derivative” will be understood as the derivative of the derivative. That is, it is a measure of the curvature of the signal, or the rate of change of the slope of the signal. It will be apparent to the skilled person that the second derivative can be calculated by applying the first derivative calculation twice in succession.
  • the start time detection condition is determined by using a first derivative analysis.
  • the start time detection condition is determined by using a second derivative analysis.
  • the amplification start time is identified by determining the second derivative.
  • the start time detection condition is reached when:
  • a measure of the width e.g., the full width at half-maximum (FWHM)
  • integrated second derivative values the latter being equivalent to the first derivative value at the zero crossing time of (ii) exceeds a further corresponding predetermined threshold value.
  • the start time detection condition is reached if a positive going peak in the second derivative is present and has a height above a given threshold and a width or integrated area under its curve that exceeds a corresponding predetermined threshold value.
  • the start time detection condition is a defined fluorescent signal of the fluorophore, e.g., the decaying temperature dependent fluorescent dye.
  • the defined fluorescent signal is greater than or equal to a steady state florescence signal of the test mixture at the substantially isothermal conditions.
  • the start time detection condition is when the temperature of the test mixture is within a threshold range of the amplification temperature.
  • the temperature of the test mixture is determined by using a liquid temperature probe or an infrared radiation (IR) temperature sensor. In one example, the temperature of the test mixture is determined by using a liquid temperature probe. In another example, the temperature of the test mixture is determined by using an IR temperature sensor.
  • IR infrared radiation
  • the temperature of the test mixture is determined using fluorescent thermometry.
  • the temperature of the test mixture is determined using a fluorescent dye, whose fluorescence intensity is a strong function of temperature.
  • the present disclosure also provides a method of amplifying one or more nucleic acids in a sample comprising subjecting the sample to ramping for a second period of time.
  • the term “ramping” will be understood to refer to the process of increasing and decreasing the temperature of a test mixture at a defined rate.
  • the present disclosure provides a method of quantitative isothermal nucleic acid amplification comprising ramping the block, and hence test mixture temperature, up and down over a range of temperatures beyond the optimal temperature range for the reaction.
  • the block temperature is ramped up and down from a first temperature and a second temperature, which are outside the optimal reaction range. At the first and second temperatures there is no amplification and the reaction temperature range is calculated to ensure that the maximum and minimum temperature is outside of the range of the optimal amplification temperature range.
  • the method comprises subjecting the test mixture to ramping for a second period of time, wherein the ramping comprises a first temperature and a second temperature.
  • the first temperature is less than an amplification temperature effective to cause the amplification reaction
  • the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature.
  • an amplification range and a constant rate of transition between the ramping up and a constant rate of transition between the ramping down is used.
  • the ramping is repeated or transitioned or cycled through in a constant manner. It will be apparent to the skilled person that this achieves heating of the amplification liquid temperature in a manner corresponding to the heating block being ramped up and down.
  • the first temperature is 2 to 5 degrees Celsius below the amplification temperature effective to cause the amplification reaction.
  • the first temperature is 2 to 5 degrees Celsius below the optimal temperature.
  • the second temperature is 2 to 5 degrees Celsius above the amplification temperature effective to cause the amplification reaction.
  • the second temperature is 2 to 5 degrees Celsius above the optimal temperature.
  • the period of ramping between the first temperature and the second temperature is between 5 and 50 seconds.
  • the period of ramping between the first temperature and the second temperature is about 5 seconds, or 10 seconds, or 15 seconds, or 20 seconds, or 25 seconds, or 30 seconds, or 35 seconds, or 40 seconds, or 45 seconds, or 50 seconds.
  • the period of ramping between the first temperature and the second temperature is about 5 seconds.
  • the period of ramping between the first temperature and the second temperature is about 10 seconds.
  • the period of ramping between the first temperature and the second temperature is about 15 seconds.
  • the period of ramping between the first temperature and the second temperature is about 20 seconds.
  • the period of ramping between the first temperature and the second temperature is about 25 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 30 seconds. In a further example, the period of ramping between the first temperature and the second temperature is about 35 seconds. In one example, the period of ramping between the first temperature and the second temperature is about 40 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 45 seconds. In a further example, the period of ramping between the first temperature and the second temperature is about 50 seconds.
  • the method comprises subjecting the sample to one or more ramps.
  • the method comprises subjecting the sample to between 1 and 50 ramps.
  • the method comprises subjecting the sample to between 10 and 40 ramps.
  • the method comprises subjecting the sample to 10 ramps.
  • the method comprises subjecting the sample to 15 ramps.
  • the method comprises subjecting the sample to 20 ramps.
  • the method comprises subjecting the sample to 25 ramps.
  • the method comprises subjecting the sample to 30 ramps.
  • the method comprises subjecting the sample to 35 ramps.
  • the method comprises subjecting the sample to 40 ramps.
  • the method comprises subjecting the sample to 50 ramps.
  • the amplification is for a duration of between about 30 minutes and 90 minutes.
  • the amplification is for a duration of about 30 minutes, or about 40 minutes, or about 50 minutes, or about 60 minutes, or about 70 minutes, or about 80 minutes or about 90 minutes. It will be apparent to the skilled person that the total time duration of the amplification will be dependent on several factors including the fluorescence detection rate, ramping time period, number of ramps and full amplification length.
  • the present disclosure provides a method of detecting one or more nucleic acids in a sample comprising: (i) ramping the temperature of the test mixture between a first temperature and a second temperature for a second period of time once the test mixture has reached the amplification temperature, wherein the temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature; and
  • the at least one detection reagent is at least one fluorophore.
  • the fluorescence emitted by the fluorophore varies with nucleic acid binding.
  • Fluorophores suitable for use in the present disclosure for detection of one or more nucleic acids will be apparent to the skilled person and/or described herein.
  • Exemplary fluorophores include, without limitation, 1,5 IAEDANS; 1,8-ANS; 4-
  • the at least one detection reagent is a fluorescent semiconductor nanocrystal or an enzyme.
  • the at least one detection reagent is a fluorescent semiconductor nanocrystal (as described, for example, in US 6,306,610).
  • the at least one detection reagent is an enzyme.
  • the enzyme is a horseradish peroxidase (HRP), an alkaline phosphatase (AP) or 0- galactosidase.
  • detection reagents facilitate the detection of a primer, for example, an amplification product produced using the primer.
  • Methods for producing/synthesizing a primer suitable for use in the present disclosure as well as methods for producing such a labeled primer are known in the art.
  • commercial sources for the production of a labeled primer are known to the skilled artisan, e.g., Sigma-Genosys, Sydney, Australia.
  • the detection time is the time at which the amplification produces sufficient fluorescence above the background signal.
  • the present disclosure also provides a method of quantifying one or more nucleic acids in the sample.
  • the method comprises determining an amplification duration for the at least one or more nucleic acids in the sample based on the detection point and the amplification start time, wherein the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
  • the method comprises determining the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids; wherein the duration of time or the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids is indicative of the quantity of the one or more nucleic acids in the sample.
  • the term “quantity” with reference to the one or more nucleic acids in the sample will be understood to refer to an amount of nucleic acid molecules. It will be apparent to the skilled person that this term encompasses both an absolute and relative value. For example, the amount may be relative to a reference or control sample. In another example, the amount may be an absolute value of the amount or quantity of nucleic acid molecules present in the sample.
  • the duration of time or the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids in the sample is determined by calculating the cross point (Cp) or detection point and comparing the Cp to a reference or control. The detection point or Cp will be reached as the amplification produces sufficient fluorescence above the background signal.
  • references for use in the methods of the present disclosure will be apparent to the skilled person and/or described herein.
  • the reference may be an internal reference (i.e., from the same subject), from a normal individual or an established data set (e.g., matched by age, sample type etc).
  • the reference is an internal reference or sample.
  • the reference is an autologous reference.
  • the internal reference is obtained from the subject at the same time as the sample under analysis. In another example, the internal reference is obtained from the subject at an earlier time point as the sample under analysis.
  • normal individual shall be taken to mean that the subject is selected on the basis that they are not known to be suffering from a disease or condition.
  • the reference is an established data set or standard. Established data sets suitable for use in the present disclosure will be apparent to the skilled person.
  • a reference is not included in an assay. Instead, a suitable reference is derived from an established data set previously generated. Data derived from processing, analyzing and/or assaying a test sample is then compared to data obtained for the sample.
  • a reference is a control sample of known quantification level (or concentration) of the nucleic acid under analysis. For example, the control sample is used to check or calibrate the quantification algorithm.
  • the type and size of the biological sample will depend upon the detection means used.
  • an assay such as, for example, PCR may be performed on a sample comprising a single cell, although a population of cells are preferred.
  • sample refers to any type of suitable material obtained from the subject.
  • the term encompasses a clinical sample, biological fluid, tissue samples, live cells and also includes cells in culture, cell supernatants, cell lysates derived therefrom.
  • the sample can be used as obtained directly from the source or following at least one-step of (partial) purification. It will be apparent to the skilled person that the sample can be prepared in any medium which does not interfere with the method of the disclosure.
  • the sample comprises cells or tissues and/or is an aqueous solution or biological fluid comprising cells or tissues.
  • Pre-treatment may involve, for example, diluting viscous fluids. Treatment of a sample may involve filtration, distillation, separation, concentration.
  • the biological sample has been derived previously from the subject. Accordingly, in one example, a method as described herein according to any embodiment additionally comprises providing the sample.
  • the sample comprising the one or more nucleic acids.
  • a method as described herein according to any embodiment is performed using an extract from a biological sample, such as, for example, DNA, mRNA or cDNA.
  • Figs. 6 to 9 show an apparatus 100, such as a nucleic acid amplification and/or detection apparatus, according to some embodiments.
  • the apparatus 100 includes measurement components configured to measure one or more characteristics of the nucleic acids within reaction vessels 102 received by the apparatus 100.
  • a sample of interest can be divided into one or more reaction vessels so that the apparatus 100 can be used to amplify the nucleic acids(s) in the divided sample and to measure multiple test and control reactions, outputting the results of these multiple tests to a user, for example, via a user interface 132 or display of the apparatus 100.
  • Fig. 7A-D show the apparatus 100 including a support 104 configured to receive respective reaction vessels 102.
  • the support 104 may have one or a plurality or apertures 106 disposed therein, each configured to receive and retain or support a respective reaction vessels 102, such as a tube.
  • the support 104 may be an elongate support with a series of apertures disposed along its length.
  • the support 104 may be composed of a material having a high thermal conductivity, for example, aluminium or copper.
  • the apparatus 100 may comprise a lid or sample receiving panel 108 configured to selectively transition between an open state and a closed state. In the open state, as shown in Figs. 7B to 7D, the support 104 and any reaction vessels 102 provided therein are accessible, whereas in the closed state, the support 104 and any reaction vessels 102 provided therein are inaccessible and closed off from the external environment, for example.
  • the circuit board 110 may comprise or connect to heating elements 120, such as resistive heater elements, or resistors.
  • the heating elements 120 are arranged or configured to heat, or transfer heat, to the heater block 124, support 104, the reaction vessel 102 supported by the heater block 124 or support 104 and/or the mixture within the reaction vessel 102.
  • the heating elements 120 may be mounted within the circuit board 110.
  • the circuit board 110 may comprise or connect to one or more temperature sensors 122.
  • the temperature sensor(s) 122 are arranged or configured to detect the temperature of the heater block 124, support 104, the reaction vessel 102 supported by the heater block 124 or support 104 and/or the mixture within the reaction vessel 102.
  • the temperature sensor(s) 122 are embedded in or otherwise attached to the heater block 124 or support 104.
  • non-contact temperature sensors 122 can be used.
  • a non-contact optical or infra-red temperature sensor such as the Melexis MLX90615 Infra-Red Thermometer senor are used in some embodiments.
  • multiple temperature sensors 122 with different characteristics are used to optimise the temperature control strategy for rapid heat up and transitions combined with good steady state temperature accuracy.
  • air flow and/or Peltier cell elements 116 are used to actively cool the support 104 to provide a rapid temperature transition to lower temperatures.
  • the apparatus 100 may include a heater block 124 configured to be heated or receive heat from the heating elements 120.
  • the heater block 124 may form at least part of the support 104 and accordingly may transfer heat to reaction vessels retained within the support 104.
  • the heater block 124 may be distinct from the support 104 but may nonetheless be configured or positioned such that heat transfer to the reaction vessels 102 retained within the support 104 can be achieved.
  • the heater block 124 may be composed of a conductive material, such as aluminium, and the support 104 may comprise wells for reaction tubes, which may be plastic.
  • the apparatus 100 may comprise a frame (not shown) to support the components of the apparatus 100.
  • the frame (not shown) may be made of a plastic moulded frame.
  • the apparatus 100 may include a fan 112 for force air cooling.
  • the apparatus 100 may include local power control for fan cooling and/or Peltier cells 116, for example, and as shown in Fig. 8 and Fig. 9B.
  • the Peltier cells 116 act as a heat pump to take heat out of or pump heat into the support 104 or heater block 124, depending on the desired temperature to be achieved for the reaction vessel and the content therein.
  • the apparatus 100 may include a heat sink 118, which may made of aluminium or copper for example, and may be exposed to forced air flow from the fan 112.
  • the apparatus 100 may comprise a light source 126 configure to emit light at one or more wavelengths, each of the one or more wavelengths being sufficient to cause stimulation of at least one respective fluorophore which may be provided within the reactive vessel(s) 102.
  • the light source 126 may be located relative to the reactive vessel(s) 102, or support 104 or heater block 124 retaining the reactive vessel(s) 102, such that light emitted from the light source 126 is directed onto the samples in the reactive vessel(s) 102.
  • the apparatus 100 may also comprise one or more fluorescence detectors 128, each configured to detect a respective wavelength range so as to detect fluorescence of a respective fluorophore.
  • the apparatus 100 comprises a controller 130.
  • the controller 130 comprises one or more processors (not shown) and memory (not shown).
  • the memory may comprise instructions, which when executed by the processor(s) cause the controller 130 to perform as a temperature controller for monitoring the temperature in the heater block 124 or support 104, and accordingly the temperature of reaction vessel(s) 102 provided thereon or therewithin.
  • the controller 130 may be configured to send instructions to the heating element(s) 120 to cause the heating elements 120 to increase the temperature of the heater block 124 or support 104.
  • the controller 130 may also be configured to receive readings or temperature information from the temperature sensor(s) 122.
  • the memory (not shown) of the controller 130 may store one or more desired temperature set points at which the controller 130 may be configured to cause the mixture of the reaction vessel to reach or maintain.
  • the desired set points may be selected by a user (not shown) interacting with the apparatus 100, for example, via user interface 132 (which may for example, be a touch screen).
  • the desired temperature or a selection of a preset temperature may be determined via instructions received from a remote device or system.
  • the controller 130 may communicate with other systems and devices via a communications interface 134, such as a USB or ethernet connection, for example.
  • the feedback loop facilitated by the controller 130, the heating element(s) 120 and the temperature sensor(s) 122 enables the temperature of the heater block 124 and/or support 104 to be controlled.
  • both the heating element(s) 120 and the temperature sensor(s) 122 are connected to electronic circuits such as the circuit board 110 and are controlled by a microprocessor or analogue control circuit such as controller 130
  • accurate temperature control strategies can be implemented.
  • proportional, integral, differential (PID) control or fuzzy logic can be used to accurately drive and stabilise the support 104 or heater block 124, and accordingly the content of reaction vessels 102 supported thereby, at a desired temperature set point.
  • this gap may be filled with air or a thermally conductive fluid such as a thermally conductive grease or silicon oil retained within the gap by a seal.
  • a desired decrease in temperature or downward ramp in temperature of the mixture in the reaction vessel 102 can be achieved via either passive cooling where the temperature difference between the reaction temperature and the ambient temperature causing cooling or active cooling, for example, where a Peltier cell is incorporated, and may be activated/deactivated in response to instructions from the controller 130.
  • the memory (not shown) comprises instructions, which when executed by the processor(s) cause the controller 130 to cause stimulation of one or more fluorophore which may be provided within the reactive vessel(s) 102.
  • the controller 130 may communicate with the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate fluorophores.
  • fluorescence as a detection signal can provide good sensitivity, and where the measurement components include multiple fluorescence detectors 128 configured to detect respective non-overlapping wavelength ranges so as not to interfere with one another, multiple channels of test and/or control reactions can be incorporated within a single reaction vessel 102.
  • the measurement components can be configured to measure optical absorption, reflection, luminance output, and/or fluorescence.
  • additional openings in the support 104 are provided to receive calibration or reference samples with specific optical characteristics. This can be used to self-calibrate or self-test the apparatus 100 during power up or measurement cycles by comparing the measured reference values against known values for the reference target.
  • the support 104 or heating block can be coupled to a vibrating mechanism or actuator such as an electromagnetic coil and slug. Actuation of this component can induce vibration in the reaction vessels mounted in the support.
  • the excitation frequent can be in the range of Hz up to kHz. Where the excitation is above 20 kHz, it can be referred to as ultrasonic mixing.
  • a piezoelectric actuator can be used.
  • the apparatus 100 includes an ultrasonic transducer configured so that the ultrasonic transducer can contact it through an opening in the support 104, with the ultrasonic transducer slightly lifting the reaction vessel so that it is not fully supported by the support 104. This may for allow efficient ultrasonic excitation of each reaction vessel only (i.e., without or with reduced excitation of the support itself) while otherwise allowing the reaction vessel 102 to be seated in good thermal contact at other rotation positions and associated measurement stations.
  • the apparatus 100 includes a barcode reader 136, for example an RFID reader or image sensor.
  • the support 104 may be configured to position a selected reaction vessel or associated disposable plastic assembly carrying or forming the vessel such that an attached label or feature is positioned in front of the barcode reader, RFID reader or image sensor. Where an image sensor is used, this can also be employed to confirm that the sample and reaction vessel or the disposable assembly that carries the reaction vessels have the correct reagents added, are assembled correctly, and are functional.
  • Figure 14 is a block diagram of an example system 200 for quantifying one or more nucleic acids in a sample.
  • the sample may form part of a test mixture provided in a reaction vessel 102 of the apparatus 100.
  • the test mixture may comprise the sample containing the one or more nucleic acids and a reagent mixture.
  • the test mixture further comprises at least one detection reagent, such as a fluorophore.
  • the system 200 comprises one or more processors 202 and memory 204.
  • the processor(s) 202 may comprise one or more microprocessors, central processing units (CPUs), application specific instruction set processors (ASIPs), application specific integrated circuits (ASICs) or other processors capable of reading and executing instruction code.
  • the memory 204 may comprise one or more volatile or non-volatile memory types.
  • the memory 204 may comprise one or more of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM) or flash memory.
  • the memory 204 is configured to store program code accessible by the processor(s) 202.
  • the program code comprises executable program code modules.
  • memory 204 is configured to store executable code modules configured to be executable by the processor(s) 202.
  • the executable code modules when executed by the processor(s) 202 cause the system 200 to perform certain functionality, as described in more detail below.
  • Memory 204 comprises data 218 and program code 220.
  • Data 218 comprises one or more of: first data set 222, second data set 224 and calibration data set 226.
  • Program code 220 comprises one or more of: a temperature control module 206, an amplification detection point module 208, an amplification process start time determination module 210, and amplification duration determination module (or quantification module) 212.
  • the program code or code modules of memory 204 which when executed by the processors 202 cause the system 202 to perform the methods of any one of Figures 15 to 18, may be stored instead in memory of the controller 130 of the apparatus 100, and the controller 120 may instead be configured to perform the functionality.
  • the methods of any one of Figures 15 to 18 may be performed by the system 200 and the controller 130 acting together, with some of the functionality being performed by the system 200 and some of the functionality being performed by the controller 130 of the apparatus 100.
  • the system 200 may comprises a network interface 214 and/or communications module 218 to facilitate communications with other systems, devices, and/or databases, for example, across a communications network (not shown), such as local area network(s) or wide area network(s), for example, the Internet.
  • the network interface 214 may facilitate communications between the system 200 and the controller 130 of the apparatus 100.
  • the communications module 216 and/or network interface 214 may comprise a combination of network interface hardware and network interface software suitable for establishing, maintaining and facilitating communication over a relevant communication channel.
  • the communications module 216 and/or network interface 214 may allow for wired or wireless communication between the system 200 and external devices, such as the controller 130 of the apparatus 100, and may use WiFi, USB, Bluetooth, or other communications protocols.
  • Figure 15 is a process flow diagram of a method 1500 of quantifying one or more nucleic acids in a sample, according to one embodiment.
  • the sample forms part of a test mixture comprising the sample, a reagent mixture and at least one detection reagent.
  • the test mixture is provided within a reaction vessel 102 arranged to be received by the apparatus 100, for example, by support 104.
  • the method 1500 may be implemented by the processor(s) 202 of system 200 executing instructions stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
  • the test mixture is subjected to substantially isothermal conditions for a first period of time.
  • the temperature control module 206 of the controller 130 may send instructions to the heating element(s) 120 to cause the heating elements 120 to increase the temperature of the heater block 124 or support 104 and receive measurements from the temperature sensor(s) 122 to monitor the temperature of the test mixture until a set point temperature or within a range of a set point temperature (as stored in memory) which corresponds with substantially isothermal conditions is achieved.
  • the set point may be an initial isothermal temperature.
  • the controller 130 responsive to the controller 130 detecting that the apparatus 100 has received the reaction vessel 102 (for example, the reaction vessel 102 is detected, for example, via a sensor, in the support 104), the controller 130 causes heating of the text mixture.
  • various different actions may cause the controller 130 to heat the text mixture to the set point. Such actions may include, for example, detection by the controller 130 of a user activating a user- selectable activator to cause the amplification process to begin, detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus, or detection by the controller 130 of the vessel receiving a reaction liquid.
  • the controller 130 records data indicative of the temperature of the test mixture over the first period of time.
  • the first period of time spans from before the test mixture is heated to a time after the test mixture reaches an amplification temperature.
  • the first period of time spans from a time when the test mixture is at an initial isothermal temperature for an initial isothermal time period.
  • the controller 130 may begin to record data indicative of the temperature once the controller 130 detects that the temperature of the test mixture is determined to be at the initial isothermal temperature.
  • the controller 130 records data indicative of the temperature of the test mixture from an initial isothermal temperature for an initial isothermal time period.
  • the initial isothermal temperature may be between 58°C and 66°C, for example about 65°C.
  • the first period of time may include the initial isothermal time period.
  • the first period of time may be substantially the same duration as the initial isothermal time period. In one example, the first period of time may be between 1 and 2 minutes.
  • the initial isothermal time period may be between 1 and 2 minutes.
  • the data indicative of the temperature of the test mixture over the first time period may be stored in a first data set.
  • the controller 130 may store the first data set locally in memory and/or may communicate it to the system 200 to be stored in memory 204.
  • the data indicative of the temperature of the test mixture over the first time period is temperature data.
  • the controller 130 may receive and record temperature data from the temperature sensor(s) 122.
  • the temperature data may be time series data.
  • the temperature data may comprise temperature measurements or readings, with each temperature measurement being associated with a time stamp indicative of the time at which the reading was obtained during the first period of time.
  • the temperature readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
  • the at least one detection reagent comprises one or more first fluorophores which vary with temperature.
  • the first fluorophore may be a decaying temperature dependent fluorescent dye, such as Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
  • HEX Hexachloro-fluorescein
  • RhB Rhodamine B
  • FAM 6-fluorescein
  • ROX carboxyrhodamine
  • the data indicative of the temperature may be first fluorescence data indicative of fluorescence emitted by the first fluorophore(s).
  • the controller 130 may be configured to cause stimulation of the first fluorophore(s) and monitor the resulting fluorescence.
  • the controller 130 may transmit instructions to the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate the first fluorophore(s).
  • the controller 130 monitors the emitted fluorescence by receiving first fluorescence data from the fluorescence detector(s) 128.
  • the controller 130 records the first fluorescence data.
  • the first fluorescence data may be time series data.
  • the first fluorescence data may comprise fluorescence measurements or readings, with each fluorescence measurement being associated with a time stamp indicative of the time at which the reading was obtained during the first period of time.
  • the fluorescence readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
  • the controller 130 may cause stimulation of the first fluorophore(s) in response to an action occurring.
  • Such actions may include: (i) detection by the controller 130 of a user activating a user-selectable activator to cause the amplification process to begin; (ii) detection by the controller 130 of the vessel being placed in a vessel support of the apparatus; (iii) detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus; (iv) detection by the controller 130 of the vessel receiving a reaction liquid; or (v) detection by the controller 130 of heat being applied to the vessel or test mixture within the vessel.
  • the controller 130 causes ramping of the temperature of the test mixture between a first temperature and a second temperature for a second or ramping period of time. The temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved.
  • the controller 130 may be configured to ramp the temperature of the test mixture between the first and second temperatures by transmitting instructions to the heating elements 120, activating air flow and/or Peltier cell elements 116 to actively cool the test mixture, for example, by cooling the support 104, and monitoring the temperature based on readings from the temperature sensor(s) 122.
  • the amplification temperature may be between 20 and 65 degrees Celsius.
  • the amplification temperature may be about 40 degrees Celsius or at about 65 degrees Celsius.
  • the first temperature is ramped for a period of approximately 100 seconds and/or the second temperature is ramped for a period of approximately 90 seconds.
  • the ramping rate may be between 0.01 degrees Celsius per second and 5 degrees Celsius per second.
  • the first temperature is less than an amplification temperature effective to cause the amplification reaction
  • the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature.
  • a temperature difference between the first and second temperatures may be between 2 and 40 degrees Celsius.
  • the first temperature may be about 25 degrees Celsius and the second temperature is about 40 degrees Celsius, and the isothermal temperature is about 40 degrees Celsius.
  • the first temperature may be about 50 degrees Celsius and the second temperature is about 65 degrees Celsius, and the isothermal temperature is about 65 degrees Celsius.
  • the controller 130 records detection reagent data associated with the test mixture during a third period of time.
  • the third time period may correspond with, or be a subset of, the second or ramping period of time.
  • the amplification detection point module 208 determines one or more detection points based on detection reagent data recorded during the third period of time. Each detection point is indicative of sufficient amplification to allow for detection of a respective nucleic acid in the sample. The detection point may be indicative of the amplification time.
  • the detection reagent(s) may be at least one second fluorophores where fluorescence emitted by the second fluorophore(s) varies with nucleic acid binding.
  • the detection reagent data may comprise fluorescence data indicative of fluorescence emitted by the second fluorophore(s) during the second period of time.
  • the second fluorophore(s) may be the first fluorophore(s); that is the first fluorophore(s) used in the above embodiment (where data indicative of the of the temperature is fluorescence data indicative of fluorescence emitted by the first fluorophore(s)) is again used as the second fluorophore(s).
  • the second fluorophore(s) may be separate fluorophore(s) to the first fluorophore(s) and may be the same type of fluorophore or a different type of fluorophore.
  • the second fluorophore may be a decaying temperature dependent fluorescent dye, such as Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
  • HEX Hexachloro-fluorescein
  • RhB Rhodamine B
  • FAM 6-fluorescein
  • ROX carboxyrhodamine
  • the controller 130 may be configured to cause stimulation of the second fluorophore(s) and to monitor the resulting fluorescence over the third time period. For example, the controller 130 may transmit instructions to the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate the second fluorophore(s).
  • the controller 130 monitors the emitted fluorescence by receiving second fluorescence data from the fluorescence detector(s) 128.
  • the controller 130 records the second fluorescence data.
  • the second fluorescence data may be time series data.
  • the second fluorescence data may comprise fluorescence measurements or readings, with each fluorescence measurement being associated with a time stamp indicative of the time at which the reading was obtained during the third period of time.
  • the fluorescence readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
  • the controller 130 may cause stimulation of the second fluorophore(s) in response to an action occurring.
  • Such actions may include: (i) detection by the controller 130 of a user activating a user-selectable activator to cause the amplification process to begin; (ii) detection by the controller 130 of the vessel being placed in a vessel support of the apparatus; (iii) detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus; (iv) detection by the controller 130 of the vessel receiving a reaction liquid; (v) detection by the controller 130 of heat being applied to the vessel or test mixture within the vessel; (vi) detection by the controller 130 of the test mixture being at a predetermined temperature, such as the amplification temperature; or (vii) determination that an initial isothermal time period has passed.
  • the memory of the apparatus 100 comprises the amplification detection point module 208 and the processor of the apparatus 100 executes the module 208 to cause the controller 130 to determine the amplification detection point(s).
  • the amplification detection point module 208 is stored remote from the apparatus 100, for example in memory 204 of system 200.
  • the data indicative of fluorescence emitted by the second fluorophore(s) during the ramping period of time is provided to the system 200.
  • the controller 130 may be configured to transmit the data to the system 200. The data may be transmitted as the second data set once all readings have been obtained, or the data may be streamed as it is being received by the controller 130 or transmitted in chunks as the chunks are collated, with each chunk comprising a predetermined number of readings.
  • the amplification process start time determination module 210 determines a start time of the amplification process based on the first data indicative of the temperature of the test mixture over the first time period.
  • the memory of the apparatus 100 comprises the amplification process start time determination module 210 and the processor of the apparatus 100 executes the module 210 to cause the controller 130 to determine the start time of the amplification process.
  • the amplification process start time determination module 210 is stored remote from the apparatus 100, for example in memory 204 of system 200.
  • the data indicative of the temperature of the test mixture over the first time period set is provided to the system 200.
  • the controller 130 may be configured to transmit the data to the system 200. The data may be transmitted as the first data set once all readings have been obtained, or the data may be streamed as it is being received by the controller 130 or transmitted in chunks as the chunks are collated, with each chunk comprising a predetermined number of readings.
  • the amplification duration determination module 212 determines the amplification duration for the at least one or more nucleic acids in the sample based on the detection point and the amplification start time.
  • the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
  • the controller 130 and/or system 200 provides the determined amplification duration(s) for respective nucleic acid(s) in the sample to the user interface 132 or display of the apparatus 100 for presentation to a user.
  • the memory of the apparatus 100 comprises the amplification duration determination module 212 and the processor of the apparatus 100 executes the module 212 to cause the controller 130 to determine the amplification duration. If the amplification process start time determination module 210 is not executing locally on the apparatus, the system 200 transmits the amplification process start time to the controller 130.
  • the amplification duration determination module 212 is stored remote from the apparatus 100, for example in memory 204 of system 200.
  • the amplification start time is provided to the system 200, if the amplification process start time determination module 210 is not executing locally on the system 200.
  • the determined detection point(s) of nucleic acid(s) in the sample are provided to the system 200, if the amplification detection point module 208 is not executing locally on the system 200.
  • the controller 130 may be configured to transmit the determined amplification start time and/or detection point(s) to the system 200.
  • One method 1700 of determining the amplification duration is described in more detail below with reference to Figure 17.
  • step 1504 is omitted, and at 1514, the amplification duration determination module 212 determines the amplification duration for the at least one or more nucleic acids in the sample based on the detection point. In other words, there is no determination of amplification process start time as discussed in more detail in Figure 16.
  • step 1506 is omitted (i.e., there is no ramping between two temperatures), and instead the amplification temperature is maintained.
  • Figure 16 is a process flow diagram of a method 1600 of determining an amplification process start time, according to some embodiments.
  • the method 1600 may be implemented by the processor(s) 202 of system 200 executing instructions (such as the amplification process start time determination module 210) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification process start time determination module 210) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
  • the amplification process start time determination module 210 determines data indicative of the temperature of the test mixture over the first time period.
  • the data may comprise the data recorded by the controller 130 at 1504 of method 1500 of process flow diagram Figure 15.
  • the amplification process start time determination module 210 is stored and executed at system 200 and the system 200 is configured to receive the data from the controller 130, as a first data set, in streamed format, or in chunks of data, as described above. It will be appreciated that method 1600 can execute while data is still being recorded and/or transmitted to the amplification process start time determination module 210.
  • the data is temperature data and in some embodiments the data is fluorescence data.
  • the amplification process start time determination module 210 determines whether a threshold data acquisition time has elapsed.
  • the threshold data acquisition time may be a period of time considered sufficient for the test mixture to be at temperature and stable.
  • the amplification process start time determination module 210 determines a start time of the amplification process as being a time at which a start time detection condition is met.
  • the amplification process start time determination module 210 determines the start time as being a time at which the temperature of the test mixture is within a threshold range of the amplification temperature.
  • the amplification process start time determination module 210 determines the start time as being a time at which a fluorescence level of the of the first fluorophore of the first fluorophore data meets a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature.
  • the amplification process start time determination module 210 determines the start time as being a time at which there is a maximum rate of change of gradient of the first fluorophore of the first fluorophore data.
  • the amplification process start time determination module 210 determines the start time as being a time at which a steady state emission level of fluorescence of the first fluorophore is achieved and is indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids.
  • the amplification process start time determination module 210 plots a graph of fluorescence against time using the third data set and determines the start time from the plotted graph.
  • the amplification process start time determination module 210 provides the determined start time to the amplification duration determination module 212.
  • Figure 17 is a process flow diagram of a method 1700 of determining one or more detection points indicative of sufficient amplification to allow for detection of one or more respective nucleic acids in the sample, according to some embodiments.
  • the method 1700 may be implemented by the processor(s) 202 of system 200 executing instructions (such as the amplification detection point module 208) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification detection point module 208) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
  • the amplification detection point module 208 determines second fluorescence data indicative of fluorescence emitted by the second fluorophore(s) during the second or ramping period of time as captured while the test mixture was being ramped between a first temperature and a second temperature at 1506 of method 1500, as described above.
  • the amplification detection point module 208 is stored and executed at system 200 and the system 200 is configured to receive the second fluorescence data from the controller 130, as a second data set, in streamed format, or in chunks of data, as described above. It will be appreciated that method 1700 can execute while data is still being recorded and/or transmitted to amplification detection point module 208.
  • the amplification detection point module 208 interpolates and/or extrapolates the second fluorescence data.
  • the amplification detection point module 208 determines a background fluorescence level for the assay before significant amplification occurs.
  • the amplification detection point module 208 provides each determined detection point(s) to the amplification duration determination module 212.
  • Figure 18 is a process flow diagram of a method 1800 of quantifying one or more nucleic acids in the sample, according to some embodiments.
  • the method 1800 may be implemented by the processor(s) 202 of system 200 executing instructions (such as amplification duration determination module 212) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification duration determination module 212) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
  • the amplification duration determination module 212 determines the determined detection point(s) and the determined start time.
  • the amplification duration determination module 212 determines an amplification duration for each of the nucleic acid(s) based on respective determined detection point(s) and the start time.
  • the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
  • the amplification duration for each of the nucleic acid(s) is calculated as the difference between the respective detection point(s) and the determined start time.
  • the amplification duration determination module 212 determines a suitable calibration dataset of known concentrations.
  • the calibration dataset may have been determined using the same quantification time determination methods as described in relation to method 1700. This calibration dataset may be saved from calibration tests run previously or run in separate tubes in the same instrument at the same time at the test in progress.
  • the calibration dataset may be stored locally to the amplification duration determination module 212, or may be retrieved from a remote database, for example.
  • the amplification duration determination module 212 determines the quantification for each nucleic acid based on the respective determined amplification duration and the calibration dataset. This amplification duration determination module 212 takes into account the dynamics of the specific isothermal amplification method (whether under ramping or not) and which may be linear, exponential or some other equation or a curve fit from experimental data.
  • the amplification duration determination module 212 provides the determined quantification for respective nucleic acid(s) in the sample to the user interface 132 or display of the apparatus 100 or system 200 for presentation to a user.
  • amplification duration determination module 212 determines an amplification duration or “Test Time” of 700 seconds, the assay type is linear in its response, and the following assay calibration data is available:
  • 600 viral copies per mL has and test time of 600. seconds
  • the amplification duration determination module 212 will interpolate the assay calibration data between the 600 second value and 800 second values, and determine a quantification of 500 viral copies per mL.
  • diagnostic testing particularly relating to a compact portable test instrument suitable for use in medical diagnostic at the Point-of-Care (POC) and in Physician’s Office Laboratories (POL).
  • POC Point-of-Care
  • POL Office Laboratories
  • the described embodiments of the present invention include nucleic acid amplification and detection apparatus that are configured to receive only one or two measurement tubes contained within a single consumable assembly and are therefore suitable for portable, point of care, or other field applications.
  • the described heated support/rotor arrangements enable multiple measurements, self-calibration and mixing functions to be performed with respect to a small number of reaction vessels or test tubes that are contained within in or are part of a disposable cartridge or vessel assembly. These features allow reduced complexity and make possible a compact, portable and relative low cost apparatus.
  • nucleic acid amplification and detection apparatus can in general be configured to receive any practical number of reaction vessels.
  • the present disclosure includes the following non-limiting Examples.
  • Example 1 Exemplary optimal and non-optimal amplification temperatures
  • Figure 1A depicts an example rate of amplification versus temperature curve for a hypothetical amplification configuration.
  • the optimal temperature i.e. the temperature at which amplification occurs most rapidly, is labelled tempo.
  • Other example temperatures, temp a , tempb and temp c which are lower than tempo, are also labelled.
  • the highlighted section indicates the range of temperatures at which amplification will occur based on variation in reaction conditions, and for temperatures above and below this range, for example temp a , amplification will either not occur (temp p ), or will not be detectable (temp a ).
  • This temperature range may only be a couple of degrees or could be as much as degrees, for example, between 60-70 degrees Celsius.
  • Figure IB provides representations of isothermal amplification reference curves of the amplification configuration of Figure 1A, produced with amplification liquid temperature at temp a , tempt,, tempc, and tempo. As temp a is outside the amplification temperature range, no detectable amplification occurs. Amplification occurs more rapidly where the reaction temperature is tempt,, and more rapidly again at temp c . The most rapid amplification occurs where the temperature of the amplification fluid is at the optimal temperature, tempo. Significant changes in amplification will affect the accuracy of the reference curve-based quantification.
  • Figure 2 depicts the block and amplification liquid temperatures versus time for an existing method of quantitative isothermal amplification when a predetermined non- optimal temperature is used for performing quantitative isothermal amplification.
  • the temperature for the reaction is selected to be at the at the lower end of the temperature range at which amplification will occur. Both the block temperature and the amplification liquid temperature are depicted, and both increase (or ramp up) to the selected non- optimal temperature, which is maintained as indicated by the horizonal portions of the block temperature and amplification liquid curves.
  • the optimal temperature of the reaction is 55 degrees Celsius and during the sample run the instrument is running at 58 degrees Celsius, the reaction is not running at the optimal temperature, and this will result in a different quantification when compared to standards (which are generally not run on the same day). It will be apparent to the skilled person that running standards at the sub-optimal temperature is expensive and requires increased time for testing.
  • Example 2 Exemplary nucleic acid amplification with ramping
  • Figure 3 provides an exemplary schematic of block temperature ramping.
  • tempo indicates an optimal temperature for isothermal amplification, and the range of temperatures at which amplification occur is shaded and labelled ‘Amplification temperature range’ .
  • the block temperature is ramped up and down between tempA and temps (as indicated by the triangle wave labelled ‘Block temperature’).
  • the area shaded within Figure 3A and 3B indicates the optimal temperature for the reaction and corresponds to rate of change of amplification as it corresponds to temperature (see e.g., Figure 1A).
  • Example 4 Exemplary methods of detecting and quantifying nucleic acids
  • FIG. 4 An exemplary schematic of the detection point of the one or more nucleic acids is depicted in Figure 4 which denotes the cross point (Cp) or detection point over a number of cycles.
  • the detection point or Cp will be reached as the amplification produces sufficient fluorescence above the background signal.
  • Figure 5A depicts the values for the different standard curves over time performed by isothermal amplification.
  • the standard curves represent different starting concentrations of template nucleic acids. The earlier the Cp, the higher the initial nucleic acid template within the sample.
  • the Cp of the standard curves can be alternatively plotted based on quantification time (Tq), where a sample with a lower Cp will have a lower Tq (e.g. CpA and TqA) and accordingly higher template nucleic acids, whereas a sample with a higher Cp will have a higher Tq (e.g. CpE and TqE) and accordingly low template nucleic acids.
  • Tq quantification time
  • the quantification time (Tq) can represent the total time that the amplification liquid is within the correct range.
  • Figure 5B depicts the Cp of the standard curves plotted against the known template concentrations of the standards.
  • a sample can be cross-referenced to standards and the target nucleic acid concentration calculated (CqX).
  • Example 6 Standard amplification at constant temperature and calculation of start time
  • Loop-mediated amplification was used to assess isothermal nucleic acid amplification and determination of a constant start time for quantification. Sample reactions are shown in Table 1 below. Samples were run at increasing concentrations at 65 degrees Celsius optimum running temperature in a final volume of lOOpL on an Axxin T8 instrument with beads.
  • the amplification curve rise times are bunched close together or even overlay and do not provide good quantification separation.
  • the fluorescence fluorophore HEX used in this test has fluorescence that falls with increasing temperature (decay).
  • This test demonstrated the typical florescence curve right at the start of the test where it falls to steady state.
  • This curve shape can be used to determine a constant start time, (tO) for the assay and quantification calculation.
  • the shaded region is the starting region of the curve.
  • the second method utilises the inflection point or point of maximum rate of change of gradient (C) in fluorescence temperature response to define the starting time point (using post-processed data) as the assay is approaching the running temperature (amplification temperature range).
  • the individual amplification time points were determined by several methods. As shown in Figure 12 the point at which the assay exponential curve crossed over a line that extrapolated a constant level above the number of amplifications thermal ramping curve for the assay response in the test was determined. The level increased slowly over time due to a low level of non-specific amplification and/or other effects which increased the background fluorescence of the assay curve.

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Abstract

The present disclosure relates to the field of nucleic acid amplification and detection, such as nucleic acid amplification to detect and quantify the presence one or more specific nucleic acids in a sample. The present disclosure also relates to methods of quantitative isothermal nucleic acid amplification and detection apparatus. The present disclosure further relates to quantitative nucleic acid amplification and detection apparatus and methods using temperature ramping and cycling.

Description

QUANTITATIVE NUCLEIC ACID AMPLIFICATION AND DETECTION APPARATUS AND METHODS THEREOF
RELATED APPLICATION DATA
This application claims priority from Australian Patent Application No. 2024900483 filed 27 February 2024 and entitled “Quantitative Nucleic Acid Amplification and Detection Apparatus and Methods Thereof’, the entire contents of which is hereby incorporated by reference.
FIELD
The present disclosure generally relates to the field of nucleic acid amplification and detection, such as nucleic acid amplification to detect and quantify the presence of one or more specific nucleic acids in a sample. The present disclosure also relates to methods of quantitative isothermal nucleic acid amplification and detection apparatus. The present disclosure further relates to quantitative nucleic acid amplification and detection apparatus and methods using temperature ramping and cycling.
BACKGROUND
Nucleic acid detection and quantification is an important part of many biomedical, environmental, veterinary, forensic and food safety processes. In particular, nucleic acid detection has become increasingly important in the discovery of genetic diseases, diagnosing pathogenic infections and monitoring disease treatment.
Nucleic acid molecules are typically present in low concentrations in biological samples, thus nucleic acid detection consists of an amplification step and a detection step. The most common amplification technique is polymerase chain reaction (PCR), which is the gold- standard for detecting nucleic acids in samples due to its reliability and specificity. PCR can amplify a single or a few copies of DNA by several orders of magnitude, generating thousands to millions of copies of the DNA sequence. Two essential components of a PCR reaction are primers containing sequences complementary to the target region and DNA polymerase. PCR relies on thermal cycling to proceed through the steps of denaturation of the double- stranded DNA (dsDNA), annealing of primers, and extension of the primer along the template by a thermostable polymerase. During this amplification process, the nucleic acid molecules double approximately each full temperature cycle, as each nucleic acid molecule is used as a template to form a new copy. Thus, the overall quantity of nucleic acids produced over a number of cycles follows an exponential curve. Almost all PCR methods use thermal cycling, or a series of heating and cooling steps and the use of a heat-stable DNA polymerase, such as Taq polymerase (originally isolated from Thermits aquatic us), which enzymatically assembles a new DNA strand using nucleotides, single-stranded DNA as a template and primers. A heating step separates the two DNA strands process (DNA melting). At a lower temperature, each strand is used as a template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Quantitative PCR (qPCR) (or also known as real-time PCR or quantitative realtime PCR) is a PCR-based technique that provides amplification and quantification of the nucleic acid species in a sample. In qPCR, fluorescence is measured after each cycle and the intensity of the fluorescent signal reflects the momentary amount of nucleic acid in the sample at that specific time. In the initial cycles, the fluorescence signal is too low to be detected from the background signal. Once a sufficient number of amplification cycles have occurred to increase the fluorescence signal above the background signal a measurement of the portion of the initial amount of template nucleic acid in the sample can be determined. However, this process relies on a high-precision thermocycler for temperature cycling and detection, which is both expensive and cumbersome in point- of-care testing settings. Additionally, there is a steep power requirement for running thermocycler units. Temperature jumps of up to 50°C may be required between successive steps of a PCR amplification reaction.
In recent years, several isothermal amplification detection techniques have been developed for detection of nucleic acids. Isothermal amplification techniques are performed at a constant temperature thereby circumventing the need for temperature cycling. The optimal temperature for isothermal amplification varies depending on the reagents used and is commonly in the range of 30-65°C. Existing isothermal amplification methods include Loop mediated amplification (LAMP), Nicking and Extension Amplification Reaction (NEAR), Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Rolling Circle Amplification (RCA), helicasedependent amplification (HDA), multiple displacement amplification (MDA), Strand displacement amplification (SDA) and recombinase polymerase amplification (RPA). However, these methods are typically only used for qualitative detection of nucleic acids (i.e., presence or absence of a nucleic acid from a specific pathogen) as accurate and reliable quantitation of nucleic acids is difficult to achieve with these methods. Quantitative detection has been applied to isothermal processes, either by conventional real-time monitoring or product accumulation or by digital end point analysis of compartmentalized parallel reactions. However, these methods have been unreliable for two reasons: (1) it is difficult to determine the starting point of the reaction as the reaction range is not taken into consideration and (2) there is variability in the conditions which results in the optimum temperature not being the same from day-to-day and from instrument-to-instrument.
Accordingly, the skilled person will appreciate from the forgoing that there is a need in the art for improved methods of amplifying, detecting and/or quantifying nucleic acids in a sample during nucleic acid amplification.
SUMMARY
In work leading up to the present invention, the inventors sought to produce a method of accurately and reliably amplifying, detecting and/or quantifying nucleic acids in a sample during nucleic acid amplification. The inventors found that when subjecting a sample to substantially isothermal conditions, they were able to accurately and reliably identify the amplification start time of the reaction by using a detection regent (e.g., a decaying temperature dependent fluorescent dye) or a threshold temperature. By determining the precise amplification start time, the inventors were able to align detected and known amplification fluorescence curves to enable accurate and consistent quantification of nucleic acids in the sample. The inventors also found that by performing ramping at a reduced ramping rate to standard thermocycling following the amplification start time, they were able to increase the time period between the amplification start time and detectable amplification curve, further increasing the accuracy of detection and quantification of nucleic acids in the sample.
The findings by the inventors provide the basis for methods of identifying an amplification start time of, amplifying, detecting and/or quantifying one or more nucleic acids in a sample.
Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) subjecting the test mixture to substantially isothermal conditions for a first period of time, wherein the first period of time spans a time from before the test mixture is heated to an amplification temperature to a time after the test mixture reaches the amplification temperature; (ii) recording, over the first time period, data indicative of the temperature of the test mixture;
(iii) determining, based on a first data set comprising the data indicative of the temperature of the test mixture over the first time period, a start time of an amplification process as being a time at which a start time detection condition is met;
(iv) ramping the temperature of the test mixture between a first temperature and a second temperature for a second period of time once the test mixture has reached the amplification temperature, wherein the temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature;
(v) recording, over a third period of time, detection reagent data associated with the test mixture, wherein the third period of time is at least a subset of the second period of time;
(vi) determining at least one detection point based on a second data set comprising the detection reagent data, wherein the at least one detection point is indicative of sufficient amplification of a respective at least one nucleic acids of the one or more nucleic acids in the sample; and
(vii) determining an amplification duration for the at least one nucleic acid in the sample based on the respective at least one detection point and the amplification start time, wherein the amplification duration is indicative of the quantity of the respect at least one nucleic acid of the one or more nucleic acids in the sample.
Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining a first data set comprising data indicative of the temperature of the test mixture over a first time period, wherein the first time period spans a time from before the test mixture was heated to an amplification temperature to a time after the test mixture reached the amplification temperature;
(ii) determining based on the first data set, a start time of an amplification process as being a time at which a start time detection condition is met; (iii) determining a second data set comprising detection reagent data associated with the test mixture and recorded during a third period of time, the third period of time being at least a subset of a second period of time during which and once the test mixture had reached the amplification temperature, the temperature of the test mixture was ramped between a first temperature and a second temperature until amplification of the at least one nucleic acid of the one or more nucleic acids in the sample was achieved, wherein the first temperature was less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature was within or exceeded an amplification temperature range comprising the amplification temperature;
(iv) determining, based on the second data set, at least one detection point indicative of sufficient amplification of a respective at least one nucleic acid of the one or more nucleic acid in the sample; and
(v) determining an amplification duration for the at least one nucleic acid in the sample based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
Some embodiments relate to a method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) monitoring a temperature of the text mixture over a first period of time;
(ii) after the temperature of the text mixture reaches the amplification temperature, ramping, at a ramping rate, the temperature of the test mixture between a first temperature and a second temperature for a second period of time, until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the ramping rate is between 0.01 degrees Celsius per second and 5 degrees Celsius per second, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature, and wherein a temperature difference between the first and second temperatures is between 2 and 40 degrees Celsius; (ii) recording, over a third time period, detection reagent data associated with the test mixture, wherein the third time period is at least a subset of the second period;
(iii) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(v) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
Some embodiments relate to a method for determining an initial quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining detection reagent data associated with the test mixture, the detection reagent data having been recorded during a third period of time once the test mixture had reached the amplification temperature, and while the temperature of the test mixture was ramped between a first temperature and a second temperature until amplification of the at least one nucleic acid of the one or more nucleic acids in the sample was achieved, wherein the ramping rate was between 0.01 degrees Celsius per second and 5 degrees Celsius per second, wherein the first temperature was less than an amplification temperature effective to cause the amplification reaction and the second temperature was within or exceeded an amplification temperature range comprising the amplification temperature, and wherein a temperature difference between the first and second temperatures is between 2 and 40 degrees Celsius;
(ii) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(iii) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
The at least one detection reagent may be at least one first fluorophore, wherein fluorescence emitted by the first fluorophore of the at least one fluorophore varies with nucleic acid binding, and wherein the detection reagent data comprises fluorescence emitted by the at least one first fluorophore of the test mixture. The at least one fluorophore may be at least one decaying temperature dependent fluorescent dye. For example, the decaying temperature dependent fluorescent dye is Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
In some embodiments, subjecting the test mixture to substantially isothermal conditions comprises heating the test mixture to a temperature of between 20 and 65 degrees Celsius. The temperature may be at about 40 degrees Celsius or at about 65 degrees Celsius.
In some embodiments,
(i) the substantially isothermal condition is at a temperature of about 40 degrees Celsius, and during ramping the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius; or
(ii)the substantially isothermal condition is at a temperature of about 65 degrees Celsius, and during ramping the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
In some embodiments, a temperature difference between the first temperature and the second temperature is between 2 and 40 degrees Celsius.
In some embodiments, during ramping:
(i) the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius; or
(ii)the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
The temperature may be ramped at a ramping rate of between 0.01 degrees Celsius per second and 5 degrees Celsius per second. During ramping the first temperature may be ramped for a period of approximately 100 seconds and/or the second temperature may be ramped for a period of approximately 90 seconds.
In some embodiments, recording, over the first time period, data indicative of the temperature of the test mixture comprises receiving temperature measurements from a sensor of the apparatus configured to sense, directly or indirectly, a temperature of the test mixture. The start time detection condition may be a threshold range of the amplification temperature. In some embodiments, the test mixture comprises at least one at least one second fluorophore that varies with temperature, and recording, over the first time period, data indicative of the temperature of the test mixture comprises determining fluorescence emitted by the at least one second fluorophore over the first time period. The at least one second fluorophore may be the at least one first fluorophore.
The start time detection condition may be a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature. The start time detection condition may be a maximum rate of change of gradient of the at least one second fluorophore. The start time detection condition may be a steady state emission level of fluorescence, the steady state emission level being indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids.
In some embodiments, the method further comprises calculating the quantity of the at least one nucleic acid based on the respective amplification duration. The amplification duration may be the time difference between the detection point and the start time. The amplification duration may be a number of ramping cycles. In some embodiments, the method further comprises comparing the amplification time duration to standards indicative of known amplification time durations for known concentrations of nucleic acids to calculate the quantity of the at least one nucleic acid.
The test mixture may further comprise a DNA polymerase with stranddisplacement activity. The DNA polymerase is selected from the group consisting of phi29 or Bsu large fragment and Bst. The test mixture may comprise an additive to lower the melting temperature (Tm) of the one or more nucleic acids in the sample, the additive may be betaine, for example. The one or more nucleic acids may be DNA or RNA. The one or more nucleic acids may be RNA and the test mixture may further comprise a reverse transcriptase.
Some embodiments relate to a method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture, and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) subjecting the test mixture to substantially isothermal conditions for a first period of time, wherein the first period of time spans a time from before the test mixture is heated to an amplification temperature to a time after the test mixture reaches the amplification temperature; (ii) recording, over the first period of time, data indicative of the temperature of the test mixture;
(iii)determining, based on a first data set comprising the data indicative of the temperature of the test mixture over the first time period, a start time of an amplification process as being a time at which a start time detection condition is met;
(iv) recording detection reagent data associated with the test mixture over a third period of time when the test mixture had reached the amplification temperature;
(v) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(vi) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
Some embodiments relate to a method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining a first data set comprising data indicative of the temperature of the test mixture over a first time period, wherein the first time period spans a time from before the test mixture was heated to an amplification temperature to a time after the test mixture reached the amplification temperature;
(ii) determining based on the first data set, a start time of the amplification process as being a time at which a start time detection condition is met;
(iii) determining a second data set comprising detection reagent data associated with the test mixture, wherein the detection reagent data was recorded during a third time period when the test mixture had reached the amplification temperature;
(iv) determining, based on the second data set, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
Some embodiments relate to a system comprising one or more processors; and memory comprising computer executable instructions, which when executed by the one or more processors, cause the system to perform any one or more of the described methods.
Some embodiments relate to a computer-readable storage medium storing instructions that, when executed by a computer, cause the computer to perform any one or more of the described methods. The computer-readable storage medium may be a nontransient computer-readable storage medium.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a series of graphical representations showing isothermal amplification measurements as a function of temperature. (A) shows the rate at which isothermal amplification occurs for different reaction temperatures and (B) shows isothermal amplification curves produced at reaction temperatures marked, where for each temperature (tempA, tempB, tempC, tempO), the same four initial target DNA concentrations are amplified.
Figure 2 is a graphical representation showing isothermal amplification when the block/amplification liquid temperature is set at a non-optimal temperature for amplification.
Figure 3 is a graphical representation showing an exemplary method of the present disclosure. (A) shows the block temperature increases (ramps up) and decreases (ramps down) between an upper temperature (tempB) and a lower temperature (tempA) (shown as solid lines) and (B) show the temperature of the reaction as a result of the ramping in block temperature cycles through the optimal temperature range. When enough cycles or ramps have occurred, sufficient amplification will allow for detection of nucleic acids in the sample (detection point).
Figure 4 is a graphical representation showing amplification fluorescence as the reaction proceeds through multiple ramps. The concentration of nucleic acids within the sample slowly increases through repeated cycles of ramps and reaches a point at which the fluorescence can be detected.
Figure 5 is a series of graphical representations showing an exemplary method of quantification in isothermal amplification. The method involves interpolating between
(A) quantification times of rate controlled isothermal amplification standard curves to
(B) calculate an unknown sample quantity of nucleic acid. Figure 6 is a block diagram showing a nucleic acid amplification and detection apparatus, according to some embodiments.
Figures 7A to D show an exemplary nucleic acid amplification and detection apparatus of the present disclosure. Figure 7A show a perspective view of the apparatus with a sample receiving panel in a closed state, Figure 7B shows a perspective view of the apparatus with the sample receiving panel in an open state and showing multiple samples received by the apparatus, Figure 7C shows the apparatus with the sample receiving panel in the open state and showing a single sample received by the apparatus and Figure 7D shows the apparatus with the sample receiving panel in the open state and showing a single sample in a closed cap tube received by the apparatus.
Figures 8A and 8B is an exploded view of an exemplary nucleic acid amplification and detection apparatus including a circuit board and with the circuit board removed, respectively.
Figure 9 is an expanded view of the heating and cooling element of the nucleic acid amplification and detection apparatus.
Figure 10 is a graphical representation showing isothermal quantification with dilutions at 10 1 to 10’6. Fluorophore is HEX, on T8 Axxin machine with beads, optimum temperature 65 degrees Celsius. Circled region shows starting region of the curve.
Figure 11 is a graphical representation showing determination of amplification start time by interpreting the fluorescence warm up curve shape. (A) Crossing point where the assay temperature crosses a defined level of fluorescence difference corresponding to temperature difference as the assay approaches the running temperature is shown by arrow A; Line at arrow B shows fixed level of fluorescence. (B) Inflection point or point of maximum rate of change of gradient in fluorescence temperate response is shown at arrow C.
Figure 12 is a graphical representation of an isothermal assay with ramp cycling. Individual amplification time points of nucleic acids at different concentrations are shown.
Figure 13 is a graphical representation showing isothermal amplification with temperature ramping. Temperature record over time using isothermal amplification at 50 degrees Celsius for 15 mins followed by temperature cycling of 100 second ramp down to 50 degrees Celsius and 90 seconds at 65 degrees Celsius.
Figure 14 is a block diagram of a system for quantifying nucleic acid in a sample, according to some embodiments.
Figure 15 is a process flow diagram of a method of quantifying one or more nucleic acids in a sample, according to one embodiment. Figure 16 is a process flow diagram of a method of determining an amplification process start time, according to a further embodiment.
Figure 17 is a process flow diagram of a method of determining one or more detection points indicative of sufficient amplification to allow for detection of one or more respective nucleic acids in the sample, according to a yet further embodiment.
Figure 18 is a process flow diagram of a method of quantifying one or more nucleic acids in the sample, according to a further embodiment.
DETAILED DESCRIPTION
General
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, for example, reference to “a” includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.
Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.
Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).
Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example but not limited to, in molecular biology and molecular genetics).
Unless otherwise indicated, sample collection and preparation and related technical techniques utilised in the present disclosure are standard procedures, well known to those skilled in the art.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.
Selected Definitions
As used herein, the term “nucleic acid” has the meaning generally understood in the art and refers to DNA or RNA or both, including any modifications.
As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone.
As used herein, “amplification” has the meaning as well-known in the art and refers to the process of making multiple copies of a nucleic acid or nucleic acid sequence.
As used herein, “amplification temperature” refers to the temperature at which an amplification enzyme initiates, or is able to initiate, amplification. The amplification temperature is a continuous variable and refers to the range of temperatures at which the amplification enzyme is active. The “optimal amplification temperature” or “optimal temperature” refers to a temperature or small temperature range at which an amplification enzyme initiates an amplification reaction at an optimum (usually fastest) rate of reaction or rate of amplification. The amplification temperature is optimised for the activity of the polymerase enzyme used in the reaction. It ensures efficient amplification of the target DNA or RNA sequence. The exact temperature depends on the specific isothermal method being used and the enzyme involved. Common isothermal amplification techniques include Loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) and Nucleic Acid Sequence Based Amplification (NASBA), each having its own optimal amplification temperature. The amplification temperature range is generally between 20-65 degrees Celsius.
As used herein, the term “amplification time” refers to the period of time required to amplify nucleic acids (DNA or RNA sequence) to a detectable level. The exact time depends on the specific isothermal method being used and the enzyme involved e.g., the activity of the polymerase enzyme used in the reaction.
As used herein, the term “period of time” shall be understood to refer to an extent of time of any length. It will be apparent to the skilled person from the disclosure herein that the first and second periods of time need not necessarily be the same and may be different.
As used herein, the term “isothermal conditions” in respect of a method of nucleic acid amplification refers to the continuous, exponential amplification of nucleic acids at a constant temperature.
As used herein, the term “substantially isothermal conditions” refers to nucleic acid amplification at a single temperature or within a narrow range of temperatures that does not vary significantly. For example, the method is carried out at a temperature that varies by only about 1-5 degrees Celsius (e.g., varying by 1, 2, 3, 4, or 5 degrees Celsius).
Methods of Quantifying Nucleic Acids
The present disclosure provides a method of quantifying one or more nucleic acids in a sample forming part of a test mixture.
Isothermal nucleic acid amplification
The present disclosure provides a method of quantifying one or more nucleic acids in a sample forming part of a test mixture, comprising subjecting the sample to substantially isothermal conditions.
In one example, the method comprises subjecting the sample to substantially isothermal conditions for a first period of time and/or a second period of time. In one example, the method comprises subjecting the sample to substantially isothermal conditions for a first period of time. In another example, the method comprises subjecting the sample to substantially isothermal conditions for a second period of time. In a further example, the method comprises subjecting the sample to substantially isothermal conditions for a first period of time and a second period of time.
The skilled person will understand that isothermal amplification comprises continuous, exponential amplification of nucleic acids at a constant temperature using enzymes, for example but not limited to strand displacing polymerases, restriction enzymes and helicases, rather than temperature changes. Isothermal amplification techniques suitable for use in the present disclosure will be apparent to the skilled person and/or described herein. Examples of known isothermal techniques include Loop- mediated Amplification (LAMP), Nicking and Extension Amplification Reaction (NEAR), Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA), Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Helicasedependent Amplification (HDA), Multiple Displacement Amplification (MDA), and Recombinase Polymerase Amplification (RPA).
Loop-mediated Amplification (LAMP) is a common isothermal amplification technique that is both sensitive and specific. LAMP employs a thermostable polymerase with strand displacement capabilities and four or more primers. The primers are designed to anneal consecutively along the target in the forward and reverse direction. Extension of the outer primers displaces the extended inner primers to release single strands. Each primer is designed to have hairpin ends that, once displaced, snap into a hairpin to facilitate self-priming and further polymerase extension. LAMP is a very fast approach to synthesising a lot of DNA, in a very short period of time. Additional loop primers can decrease the amplification time but complicates the reaction mixture. LAMP is used commercially, for example, in rapid molecular tests and microorganism detection.
Nicking and Extension Amplification Reaction (NEAR) uses two sets of primers, a strand displacing polymerase, and a restriction endonuclease. One set of primers serve to displace the initially extended second set of primers to create a single- strand for the next primer to bind. A restriction site is present in the 5' region of the primer. Key components required for NEAR are enzymes, also known as nicking endonucleases (NEases) which are enzymes derived from a mutation of restriction enzymes, and DNA polymerase. DNA polymerases with strand displacement activity include Phi 29, Klenow Fragment, vent, and Bst DNA polymerase. Among these enzymes, the most commonly used is Bst DNA polymerase. Other reactive materials are needed in this system, such as buffer mixture, dNTPs, and primers. Once all the materials are present, the two enzymes interact with each other in a suitable system and the target DNA can be amplified exponentially. When the above components are present, a suitable temperature is applied, and nicking enzyme-combined amplification begins. Regardless of the type of isothermal amplification, nicking enzyme-combined amplification includes the following four steps: (1) the nicking enzyme recognises and cleaves a specific site on a single strand in the double strand, exposing the 3' end; (2) the polymerase extends a new strand from the 3' end, the new strand containing the recognition site for the nicking enzyme; (3) the nascent strands may be used directly as a product but are more often used to participate in downstream reactions; (4) a large amount of target DNA/RNA product is obtained (Cao et al 2022). NEAR can be divided into categories based on the combination of different types of isothermal amplification with nicking enzymes, for example, typical Nicking Enzyme-Combined Amplification (EXPAR), Nicking Enzymes Combined with SDA, Nicking Enzymes Combined with RCA, and NEAR on its own.
Strand Displacement Amplification (SDA) is a continuous nicking and polymerisation/displacement process catalysed by nicking enzymes, for example, restriction enzyme Hindi, and DNA polymerases, for example, an exonuclease-deficient DNA polymerase, Hexachloro-fluorescein (HEX) or Rhodamine B (RhB). The restriction endonuclease will nick the target DNA, allowing DNA polymerase to extend the 3’ end. In the presence of primer 1, primer 2, template 1, and template 2, two primertemplate complexes are formed after denaturation. Both primers contain an identification sequence that is used to cause the identification cleavage of the nicking enzyme. After being cut, DNA polymerase extends the 3' end of the double strand to generate dsDNA containing the complete nicking site, which will be cut by a nicking enzyme to generate a new 3' end at the notch, triggering a new extension reaction and causing the displacement of the downstream target strand. The ssDNA from the primer 1 -template 1 complex can be used as a template for primer 2, and the product from the primer2- template2 complex can also be used as template for primer 1. This cycle results in exponential amplification of the target. This technique can have some limitations compared to other isothermal techniques, including lower primer specificity.
Rolling Circle Amplification (RCA) is an isothermal nucleic acid amplification method based on ligase binding, primer extension, and strand displacement amplification reaction [73], which mimics the rolling loop replication process of microbial circular DNA in nature. Under constant temperature conditions, many repetitive sequences complementary to the ring probe can be generated. After the isothermal linear amplification of the ring probe in vitro is realized in combination with the nicking enzyme, RCA can change from linear amplification to exponential amplification or multi-primer amplification. Only a segment of a nicking enzyme needs to be designed on the ring probe. With amplification, a long strand of DNA with a specific sequence interval is obtained. RCA is a linear amplification, but when nicking enzymes are used in conjunction with multiple templates, the amplification becomes exponential.
Transcription-Mediated Amplification (TMA) involves the isothermal amplification of rRNA by reverse transcription and subsequent generation of numerous transcripts by RNA polymerase. Following amplification, these RNA copies are hybridised with a complementary oligonucleotide probe for detection via a chemiluminescent tag. TMA produces 100-1000 copies per cycle, resulting in a 10 billionfold increase within 15-30 min. TMA is popular in clinical diagnostics with numerous commercial tests based on the technique available to clinical laboratories, and is also used in food and water safety, among other areas.
Nucleic Acid Sequence-Based Amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature, for example at 41 degrees Celsius. NASBA is a two-step process used to produce multiple copies of single stranded RNA by annealing specially designed primers, then utilising an enzyme cocktail to amplify it. NASBA is also commonly referred to as “self- sustained sequence replication” whereby RNA is converted into cDNA, which is transcribed to produce more RNA. NASBA routinely produces a high level of amplification, on the order to 109. NASBA has been used to develop rapid diagnostic tests for several pathogenic viruses with single- stranded RNA genomes, for example, influenza A, zika virus, foot-and-mouth disease virus, severe acute respiratory syndrome (SARS)-associated coronavirus, human bocavirus (HBoV) and also parasites like Trypanosoma brucei.
Single Primer Isothermal Amplification (SPIA) amplification is an approach using only one DNA-RNA chimeric primer along with RNase H and a DNA polymerase with strand displacement activity. This approach is capable of amplifying more DNA than PCR when primer concentrations are increased. Applications using this technique include on-site diagnosis and DNA detection.
Multiple Displacement Amplification (MDA) amplifies nucleic acid sequences using a strand-displacing DNA polymerase and multiple primer sets (Gill, & Ghaemi, 2008). Variations of this technique have been used for isothermal whole genome sequencing. One advantage of IMDA over PCR is its sensitivity and specificity. This is especially helpful when the amount of DNA in the sample is very low. Helicase-dependent Amplification (HD A) is an amplification approach that based on the in vivo DNA replication process by using helicase to unwind DNA, allowing primers to bind. To prevent single stranded DNA from associating with its complimentary strand or destabilising, two accessory proteins: MutL and single- stranded DNA-binding protein (SSB) are also used. This technique is a simple approach to isothermal amplification that has been used to develop sensitive viral and bacterial detection.
Recombinase Polymerase Amplification (RPA) amplifies DNA at a constant temperature (37-42C) using a recombinase, primers, a single-stranded DNA binding protein (SSB), and a strand displacing DNA polymerase. In this technique, the recombinase is complexed with the primer. The complex is able to bind with doublestranded DNA at homologous sequences through a strand exchange. After the exchange, a single-stranded binding protein, T4 gp32, stabilises the displaced strand. Finally, Bsu polymerase extends the primers, creating a new complete copy of the template.
As will be apparent to the skilled person, the specific primers used in a method of the present disclosure will depend on the assay format used. Methods for designing primers for, for example, isothermal amplification methods disclosed herein are known in the art and described, for example, in Dieffenbach 1995.
Furthermore, a primer (or the sequence thereof) is assessed to determine the temperature at which it denatures from a target nucleic acid (i.e., the melting temperature of the probe or primer, or Tm). The Tm is defined as the temperature at which half of the DNA strands are in the random coil or single- stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence. Methods of determining Tm are known in the art and described, for example, in Santa Lucia, 1995 or Bresslauer et al., 1986.
In one example, the sample comprises an additive to lower the Tm of the one or more nucleic acids in the sample. Examples of additives suitable for lowering Tm of nucleic acids are known in the art and include, for example, betaine and formamide. In one example, the additive is betaine.
It will be apparent to the skilled person that in isothermal amplification, DNA polymerases are used to separate duplex DNA. The rate at which isothermal amplification occurs varies depending on the temperature of the reaction fluid in which the polymerase enzyme is present. For a specific isothermal amplification configuration, there is an optimal temperature at which amplification will occur most rapidly. The optimal temperature at which amplification will occur is dependent on the reagent constituents including the specific primers and polymerases used. The variation is partially due to the instrument-to-instrument variation as well as the ambient temperature in which the reaction sample is prepared. Additionally, even if the instrument was to run the sample at the exact same temperature, the next batch of reagents may have a peak that has a different optimal temperature range. Amplification will also occur for a range of temperatures above and below this optimal temperature but at slower rates.
In one example, the sample comprises a DNA polymerase with stranddisplacement activity. For example, the DNA polymerase is selected from the group consisting of phi29, Bsu large fragment and Bst. For example, DNA polymerases phi29 or Bsu large fragment may be used in moderate temperature reactions (20-40°C) or a Bst DNA polymerase may be used for higher temperature reactions (50-65°C). Primer-based annealing and extending are then performed.
The skilled person will understand that the optimal temperature (temp0) is the temperature at which nucleic acid amplification occurs most rapidly. Amplification will occur within a temperature range above and below this optimal temperature. This temperature range may only be a couple of degrees (e.g., 2-5 degrees Celsius) or could be as much as degrees, for example, between 60-70 degrees Celsius. Small changes in the amplification liquid temperature may result in a large range in the rate at which isothermal amplification will occur. The most rapid amplification occurs where the temperature of the amplification fluid is at the optimal temperature. Significant changes in amplification will affect the accuracy of the reference curve-based quantification. For example, if the optimal temperature of the reaction is 55 degrees Celsius and during the sample run the instrument is running at 58 degrees Celsius, the reaction is not running at the optimal temperature, and this will result in a different quantification when compared to standards (which are generally not run on the same day). It will be apparent to the skilled person that running standards at the sub-optimal temperature is expensive and requires increased time for testing.
The skilled person will appreciate that the non-optimal amplification fluid temperature setting method has a number of challenges. Firstly, it relies on precise knowledge of the temperature range at which isothermal amplification will occur for a specific amplification configuration. For example, when a non-optimal temperature is selected which is close to the edge of this range, and the range is not accurately determined, there is chance that false negatives will result. Secondly, this method may require accurate amplification fluid temperature control. For example, if a non-optimal temperature is selected for performing the quantified isothermal amplification, a relatively minor error in amplification fluid temperature may lead to large variations in the rate at which isothermal amplification will occur. As repeatability is critical for creating and utilising reference curves in quantitative isothermal amplification, highly precise temperature control is required. In practice, it is difficult to accurately control amplification fluid temperature within less than 0.5 degrees Celsius accuracy, and, when designing tests, the effects of temperature errors on amplification rate must be considered.
Detection reagents
In one example of any method of the disclosure, the method comprises identifying the amplification start time when a first detection reagent is at a threshold level.
As used herein, a “detection reagent” is a substance or solution that reacts with certain other substances or substances in a characteristic manner. Typical characteristics include colour change, fluorescence, formation of a precipitation etc and suitable detection reagents are well-known in the art and/or described herein.
In one example, the first detection reagent is a temperature dependent fluorescent dye. For example, the first detection reagent is a decaying temperature dependent fluorescent dye.
The skilled person will understand that temperature dependent fluorescent dyes produce fluorescent signals that either decay or increase in response to increasing temperature. For example, where a decaying temperature dependent fluorescent dye is used, the fluorescent signal decreases as the temperature ramps up. An exemplary temperature dependent dye is Rhodamine B (RhB) that when stimulated by an excitation source of between approximately 500nm-550nm, RhB produces a fluorescent signal with an emission peak in the range of 565nm to 590nm, which diminishes as temperature increases.
In one example, the start time detection condition is:
(i) a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature;
(ii) a maximum rate of change of gradient of a fluorophore, and wherein determining the start time comprises determining the time corresponding to the maximum rate of change of gradient of the second set of fluorescence data; and/or
(iii) a time at which a steady state emission level of fluorescence is achieved and is indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids. In one example, the start time detection condition is a maximum rate of change of gradient of the fluorescent signal of a fluorophore, e.g., the decaying temperature dependent fluorescent dye.
Methods of determining a maximum rate of change of gradient of the fluorescent signal of the decaying temperature dependent fluorescent dye will be apparent to the skilled person and/or described herein.
In one example, the maximum rate of change of gradient of the fluorescent signal of the decaying temperature dependent fluorescent dye is determined by using a first derivative or a second derivative analysis. For example, the first derivative or second derivative is determined.
As used herein, the term “first derivative” will be understood to refer to the rate of change of a functions output with respect to its input, such as the rate of change of fluorescence over time (i.e., dy/dx). For example, the first derivative is the slope of the tangent to the fluorescent signal at each point.
As used herein, the term “second derivative” will be understood as the derivative of the derivative. That is, it is a measure of the curvature of the signal, or the rate of change of the slope of the signal. It will be apparent to the skilled person that the second derivative can be calculated by applying the first derivative calculation twice in succession.
In one example, the start time detection condition is determined by using a first derivative analysis.
In one example, the start time detection condition is determined by using a second derivative analysis. For example, the amplification start time is identified by determining the second derivative.
In one example, the start time detection condition is reached when:
(i) at least one of the second derivative values exceeds a predetermined positive second derivative threshold value; and
(ii) the second derivative values cross zero after having exceeded the second derivative threshold value (i), the time at which this occurs being referred to as the zero crossing time; and
(iii) a measure of the width (e.g., the full width at half-maximum (FWHM)) or integrated second derivative values (the latter being equivalent to the first derivative value at the zero crossing time of (ii)) exceeds a further corresponding predetermined threshold value.
In one example, the start time detection condition is reached if a positive going peak in the second derivative is present and has a height above a given threshold and a width or integrated area under its curve that exceeds a corresponding predetermined threshold value.
In one example, the start time detection condition is a defined fluorescent signal of the fluorophore, e.g., the decaying temperature dependent fluorescent dye.
Methods of determining the defined fluorescent signal of the decaying temperature dependent fluorescent dye will be apparent to the skilled person and/or described herein.
In one example, the defined fluorescent signal is greater than or equal to a steady state florescence signal of the test mixture at the substantially isothermal conditions.
Test mixture temperature
In one example of any method of the disclosure, the start time detection condition is when the temperature of the test mixture is within a threshold range of the amplification temperature.
Methods of determining the temperature threshold will be apparent to the skilled person and/or described herein.
In one example, the temperature of the test mixture is determined by using a liquid temperature probe or an infrared radiation (IR) temperature sensor. In one example, the temperature of the test mixture is determined by using a liquid temperature probe. In another example, the temperature of the test mixture is determined by using an IR temperature sensor.
In one example, the temperature of the test mixture is determined using fluorescent thermometry. For example, the temperature of the test mixture is determined using a fluorescent dye, whose fluorescence intensity is a strong function of temperature.
Nucleic acid amplification with ramping
The present disclosure also provides a method of amplifying one or more nucleic acids in a sample comprising subjecting the sample to ramping for a second period of time.
As used herein, the term “ramping” will be understood to refer to the process of increasing and decreasing the temperature of a test mixture at a defined rate.
The present disclosure provides a method of quantitative isothermal nucleic acid amplification comprising ramping the block, and hence test mixture temperature, up and down over a range of temperatures beyond the optimal temperature range for the reaction.
In one example, the block temperature is ramped up and down from a first temperature and a second temperature, which are outside the optimal reaction range. At the first and second temperatures there is no amplification and the reaction temperature range is calculated to ensure that the maximum and minimum temperature is outside of the range of the optimal amplification temperature range.
In one example, the method comprises subjecting the test mixture to ramping for a second period of time, wherein the ramping comprises a first temperature and a second temperature. For example, the first temperature is less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature.
The skilled person will be aware that enzymes denature at temperatures above a certain threshold and will be aware of the denaturation threshold and/or how to ascertain it for any given amplification enzyme. It will therefore be apparent to a skilled person that calculation of the maximum liquid temperature should take enzyme denaturation into account.
In one example, an amplification range and a constant rate of transition between the ramping up and a constant rate of transition between the ramping down is used. For example, the ramping is repeated or transitioned or cycled through in a constant manner. It will be apparent to the skilled person that this achieves heating of the amplification liquid temperature in a manner corresponding to the heating block being ramped up and down.
In one example, the first temperature is 2 to 5 degrees Celsius below the amplification temperature effective to cause the amplification reaction. For example, the first temperature is 2 to 5 degrees Celsius below the optimal temperature.
In one example, the second temperature is 2 to 5 degrees Celsius above the amplification temperature effective to cause the amplification reaction. For example, the second temperature is 2 to 5 degrees Celsius above the optimal temperature.
It will be apparent to the skilled person that due to the lag between block temperature and amplification liquid temperature, the amplification liquid temperature follows a more rounded waveform.
In one example, the period of ramping between the first temperature and the second temperature is between 5 and 50 seconds. For example, the period of ramping between the first temperature and the second temperature is about 5 seconds, or 10 seconds, or 15 seconds, or 20 seconds, or 25 seconds, or 30 seconds, or 35 seconds, or 40 seconds, or 45 seconds, or 50 seconds. In one example, the period of ramping between the first temperature and the second temperature is about 5 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 10 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 15 seconds. In a further example, the period of ramping between the first temperature and the second temperature is about 20 seconds. In one example, the period of ramping between the first temperature and the second temperature is about 25 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 30 seconds. In a further example, the period of ramping between the first temperature and the second temperature is about 35 seconds. In one example, the period of ramping between the first temperature and the second temperature is about 40 seconds. In another example, the period of ramping between the first temperature and the second temperature is about 45 seconds. In a further example, the period of ramping between the first temperature and the second temperature is about 50 seconds.
In one example, the method comprises subjecting the sample to one or more ramps. For example, the method comprises subjecting the sample to between 1 and 50 ramps. In one example, the method comprises subjecting the sample to between 10 and 40 ramps. For example, 10 ramps, or 15 ramps, or 20 ramps, or 25 ramps, or 30 ramps, or 35 ramps, or 40 ramps. In one example, the method comprises subjecting the sample to 10 ramps. In another example, the method comprises subjecting the sample to 15 ramps. In a further example, the method comprises subjecting the sample to 20 ramps. In one example, the method comprises subjecting the sample to 25 ramps. In another example, the method comprises subjecting the sample to 30 ramps. In a further example, the method comprises subjecting the sample to 35 ramps. In one example, the method comprises subjecting the sample to 40 ramps. In another example, the method comprises subjecting the sample to 50 ramps.
In one example, the amplification is for a duration of between about 30 minutes and 90 minutes. For example, the amplification is for a duration of about 30 minutes, or about 40 minutes, or about 50 minutes, or about 60 minutes, or about 70 minutes, or about 80 minutes or about 90 minutes. It will be apparent to the skilled person that the total time duration of the amplification will be dependent on several factors including the fluorescence detection rate, ramping time period, number of ramps and full amplification length.
Methods of Nucleic Acid Detection
The present disclosure provides a method of detecting one or more nucleic acids in a sample comprising: (i) ramping the temperature of the test mixture between a first temperature and a second temperature for a second period of time once the test mixture has reached the amplification temperature, wherein the temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature; and
(ii) detecting at least one detection reagent in the test mixture at a detection point during the ramping, wherein the presence of the detection reagent in the test mixture is indicative of the presence of the one or more nucleic acids in the sample.
In one example, the at least one detection reagent is at least one fluorophore. For example, the fluorescence emitted by the fluorophore varies with nucleic acid binding. Fluorophores suitable for use in the present disclosure for detection of one or more nucleic acids will be apparent to the skilled person and/or described herein. Exemplary fluorophores include, without limitation, 1,5 IAEDANS; 1,8-ANS; 4-
Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein (pH 10); 5 -Carboxy tetramethylrhodamine (5- TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5- Carboxy tetramethylrhodamine); 6-Carboxyrhodamine 6C; 6-CR 6G; 6-JOE; 7-Amino- 4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro- 2-methoxy acridine); Acridine Orange+DNA; Acridine Orange+RNA; Acridine Orange, both DNA & RNA; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633; Alexa Fluor 647; Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA- X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn2+; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG CBQCA; ATTO-TAG FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisamninophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET Bimane; Bisbenzamnide; Bisbenzimide (Hoechst); bis-BTC=Ratio Dye, Zn2+; Blancophor FFG; Blancophor SV; BOBO-1; BOBO-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO-1; BO-PRO-3; Brilliant Sulphoflavin FF; BTC-Ratio Dye Ca2+; BTC-5N-atio Dye, Zn2+; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP — Cyan Fluorescent Protein; CFP/YFP; FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca2+ Dye); Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C- phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2; Cy3.1 8; Cy3.5; Cy3; Cy5.1 8; Cy5.5; Cy5; Cy7; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16- ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DilC 18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC 18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescent protein; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer- 1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; FITC Antibody; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red (high pH); Fura Red/Fluo-3; Fura-2, high calcium; Fura-2, low calcium; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP), GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxystilbamidine (FluoroGold); Hydroxy tryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO- 1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LIVE/DEAD Kit Animal Cells, Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue, LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag- Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mito tracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Greene 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP; sgBFP (super glow BFP); sgGFP; sgGFP (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYT; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red; Texas Red- X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE- Cy5); TRITC (TetramethylRodamine-IsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3.
In one example, the at least one detection reagent is a fluorescent semiconductor nanocrystal or an enzyme.
In one example, the at least one detection reagent is a fluorescent semiconductor nanocrystal (as described, for example, in US 6,306,610).
In a further example, the at least one detection reagent is an enzyme. For example, the enzyme is a horseradish peroxidase (HRP), an alkaline phosphatase (AP) or 0- galactosidase.
It will be apparent to the skilled person that such detection reagents facilitate the detection of a primer, for example, an amplification product produced using the primer. Methods for producing/synthesizing a primer suitable for use in the present disclosure as well as methods for producing such a labeled primer are known in the art. Furthermore, commercial sources for the production of a labeled primer are known to the skilled artisan, e.g., Sigma-Genosys, Sydney, Australia.
In one example, the detection time (or detection point) is the time at which the amplification produces sufficient fluorescence above the background signal.
Methods of Nucleic Acid Quantification
The present disclosure also provides a method of quantifying one or more nucleic acids in the sample. For example, the method comprises determining an amplification duration for the at least one or more nucleic acids in the sample based on the detection point and the amplification start time, wherein the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
In one example, the method comprises determining the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids; wherein the duration of time or the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids is indicative of the quantity of the one or more nucleic acids in the sample. As used herein, the term “quantity” with reference to the one or more nucleic acids in the sample will be understood to refer to an amount of nucleic acid molecules. It will be apparent to the skilled person that this term encompasses both an absolute and relative value. For example, the amount may be relative to a reference or control sample. In another example, the amount may be an absolute value of the amount or quantity of nucleic acid molecules present in the sample.
In one example, the duration of time or the number of ramping cycles between the detection time and the amplification start time for the one or more nucleic acids in the sample is determined by calculating the cross point (Cp) or detection point and comparing the Cp to a reference or control. The detection point or Cp will be reached as the amplification produces sufficient fluorescence above the background signal.
Methods of determining the cross point or detection point and methods of comparing this point to a reference or control will be apparent to the skilled person and/or described herein.
References
As will be apparent from the preceding description, some assays of the present disclosure may utilise a suitable reference or control for quantification.
Suitable references for use in the methods of the present disclosure will be apparent to the skilled person and/or described herein. For example, the reference may be an internal reference (i.e., from the same subject), from a normal individual or an established data set (e.g., matched by age, sample type etc).
In one example, the reference is an internal reference or sample. For example, the reference is an autologous reference. In one example, the internal reference is obtained from the subject at the same time as the sample under analysis. In another example, the internal reference is obtained from the subject at an earlier time point as the sample under analysis.
As used herein, the term “normal individual” shall be taken to mean that the subject is selected on the basis that they are not known to be suffering from a disease or condition.
In one example, the reference is an established data set or standard. Established data sets suitable for use in the present disclosure will be apparent to the skilled person.
In one example, a reference is not included in an assay. Instead, a suitable reference is derived from an established data set previously generated. Data derived from processing, analyzing and/or assaying a test sample is then compared to data obtained for the sample. In one example, a reference is a control sample of known quantification level (or concentration) of the nucleic acid under analysis. For example, the control sample is used to check or calibrate the quantification algorithm.
Samples
As will be apparent to the skilled person, the type and size of the biological sample will depend upon the detection means used. For example, an assay, such as, for example, PCR may be performed on a sample comprising a single cell, although a population of cells are preferred.
As used herein, the term “sample” or “biological sample” refers to any type of suitable material obtained from the subject. The term encompasses a clinical sample, biological fluid, tissue samples, live cells and also includes cells in culture, cell supernatants, cell lysates derived therefrom. The sample can be used as obtained directly from the source or following at least one-step of (partial) purification. It will be apparent to the skilled person that the sample can be prepared in any medium which does not interfere with the method of the disclosure. Typically, the sample comprises cells or tissues and/or is an aqueous solution or biological fluid comprising cells or tissues. The skilled person will be aware of selection and pre-treatment methods. Pre-treatment may involve, for example, diluting viscous fluids. Treatment of a sample may involve filtration, distillation, separation, concentration.
In one example, the biological sample has been derived previously from the subject. Accordingly, in one example, a method as described herein according to any embodiment additionally comprises providing the sample. For example, the sample comprising the one or more nucleic acids.
In one example, a method as described herein according to any embodiment is performed using an extract from a biological sample, such as, for example, DNA, mRNA or cDNA.
As will be apparent from the disclosure herein, the method according to any disclosure provided herein comprises providing a sample as part of a test mixture. For example, the sample forms part of the test mixture together with other excipients, for example, at least one detection reaction and/or a reagent mixture.
Apparatus
Figs. 6 to 9 show an apparatus 100, such as a nucleic acid amplification and/or detection apparatus, according to some embodiments. To perform measurements on the sample during and/or following amplification, the apparatus 100 includes measurement components configured to measure one or more characteristics of the nucleic acids within reaction vessels 102 received by the apparatus 100. A sample of interest can be divided into one or more reaction vessels so that the apparatus 100 can be used to amplify the nucleic acids(s) in the divided sample and to measure multiple test and control reactions, outputting the results of these multiple tests to a user, for example, via a user interface 132 or display of the apparatus 100.
Fig. 7A-D show the apparatus 100 including a support 104 configured to receive respective reaction vessels 102. As illustrated in Fig. 8A, the support 104 may have one or a plurality or apertures 106 disposed therein, each configured to receive and retain or support a respective reaction vessels 102, such as a tube. For example, the support 104 may be an elongate support with a series of apertures disposed along its length. The support 104 may be composed of a material having a high thermal conductivity, for example, aluminium or copper. As shown in Figs. 7A to 7D, the apparatus 100 may comprise a lid or sample receiving panel 108 configured to selectively transition between an open state and a closed state. In the open state, as shown in Figs. 7B to 7D, the support 104 and any reaction vessels 102 provided therein are accessible, whereas in the closed state, the support 104 and any reaction vessels 102 provided therein are inaccessible and closed off from the external environment, for example.
As shown in Figures 7 to 9, the apparatus 100 may include a circuit board such as a printed circuit board (PCB) 110. The circuit board 110 may be deployed on an upper surface of the apparatus 100, and may be configured to accommodate and fit around the support 104. Electrical connections to electrical components of the apparatus 100, such as heating elements and temperature sensors may be provided by way of capable connections or slip rings.
The circuit board 110 may comprise or connect to heating elements 120, such as resistive heater elements, or resistors. The heating elements 120 are arranged or configured to heat, or transfer heat, to the heater block 124, support 104, the reaction vessel 102 supported by the heater block 124 or support 104 and/or the mixture within the reaction vessel 102. The heating elements 120 may be mounted within the circuit board 110.
The circuit board 110 may comprise or connect to one or more temperature sensors 122. The temperature sensor(s) 122 are arranged or configured to detect the temperature of the heater block 124, support 104, the reaction vessel 102 supported by the heater block 124 or support 104 and/or the mixture within the reaction vessel 102. The temperature sensor(s) deployed on, at or near the heater block 124 or support 104. In some embodiment, the temperature sensor(s) 122 are embedded in or otherwise attached to the heater block 124 or support 104.
In some embodiment, one or more of the temperature sensor(s) are non-contact temperature sensor 122, which may for example, be mounted to the circuit board 110. The temperature sensor 122 measures the temperature of support 102 or heater block 124 but is mounted to the circuit board 110. In some embodiments, the temperature sensor 122 is connected to an annular member or vane (not shown) that is disposed within an annular channel in the lower portion of the support 104 with a small gap therebetween. This arrangement provides good thermal coupling to the sensor 122 from the support 104 across the small gap in the metallic vane (not shown) attached to the sensor 122.
In some embodiments, alternative types of non-contact temperature sensors 122 can be used. For example, a non-contact optical or infra-red temperature sensor such as the Melexis MLX90615 Infra-Red Thermometer senor are used in some embodiments. In some embodiments, multiple temperature sensors 122 with different characteristics are used to optimise the temperature control strategy for rapid heat up and transitions combined with good steady state temperature accuracy. In some embodiments, air flow and/or Peltier cell elements 116 are used to actively cool the support 104 to provide a rapid temperature transition to lower temperatures.
The apparatus 100 may include a heater block 124 configured to be heated or receive heat from the heating elements 120. In some embodiments, the heater block 124 may form at least part of the support 104 and accordingly may transfer heat to reaction vessels retained within the support 104. In other embodiments, the heater block 124 may be distinct from the support 104 but may nonetheless be configured or positioned such that heat transfer to the reaction vessels 102 retained within the support 104 can be achieved. The heater block 124 may be composed of a conductive material, such as aluminium, and the support 104 may comprise wells for reaction tubes, which may be plastic.
The apparatus 100 may comprise a frame (not shown) to support the components of the apparatus 100. The frame (not shown) may be made of a plastic moulded frame. The apparatus 100 may include a fan 112 for force air cooling. The apparatus 100 may include local power control for fan cooling and/or Peltier cells 116, for example, and as shown in Fig. 8 and Fig. 9B. The Peltier cells 116 act as a heat pump to take heat out of or pump heat into the support 104 or heater block 124, depending on the desired temperature to be achieved for the reaction vessel and the content therein. The apparatus 100 may include a heat sink 118, which may made of aluminium or copper for example, and may be exposed to forced air flow from the fan 112. In some embodiments, the apparatus 100 may comprise a light source 126 configure to emit light at one or more wavelengths, each of the one or more wavelengths being sufficient to cause stimulation of at least one respective fluorophore which may be provided within the reactive vessel(s) 102. The light source 126 may be located relative to the reactive vessel(s) 102, or support 104 or heater block 124 retaining the reactive vessel(s) 102, such that light emitted from the light source 126 is directed onto the samples in the reactive vessel(s) 102. The apparatus 100 may also comprise one or more fluorescence detectors 128, each configured to detect a respective wavelength range so as to detect fluorescence of a respective fluorophore.
As illustrated in Figure 6, the apparatus 100 comprises a controller 130. The controller 130 comprises one or more processors (not shown) and memory (not shown). The memory may comprise instructions, which when executed by the processor(s) cause the controller 130 to perform as a temperature controller for monitoring the temperature in the heater block 124 or support 104, and accordingly the temperature of reaction vessel(s) 102 provided thereon or therewithin. To this end, the controller 130 may be configured to send instructions to the heating element(s) 120 to cause the heating elements 120 to increase the temperature of the heater block 124 or support 104. The controller 130 may also be configured to receive readings or temperature information from the temperature sensor(s) 122. The memory (not shown) of the controller 130 may store one or more desired temperature set points at which the controller 130 may be configured to cause the mixture of the reaction vessel to reach or maintain. The desired set points may be selected by a user (not shown) interacting with the apparatus 100, for example, via user interface 132 (which may for example, be a touch screen). In some embodiments, the desired temperature or a selection of a preset temperature may be determined via instructions received from a remote device or system. For example, the controller 130 may communicate with other systems and devices via a communications interface 134, such as a USB or ethernet connection, for example. The feedback loop facilitated by the controller 130, the heating element(s) 120 and the temperature sensor(s) 122 enables the temperature of the heater block 124 and/or support 104 to be controlled. In some embodiments, where both the heating element(s) 120 and the temperature sensor(s) 122 are connected to electronic circuits such as the circuit board 110 and are controlled by a microprocessor or analogue control circuit such as controller 130, accurate temperature control strategies can be implemented. For example, proportional, integral, differential (PID) control or fuzzy logic can be used to accurately drive and stabilise the support 104 or heater block 124, and accordingly the content of reaction vessels 102 supported thereby, at a desired temperature set point. In some embodiments, where the thermal transfer is across a gap, this gap may be filled with air or a thermally conductive fluid such as a thermally conductive grease or silicon oil retained within the gap by a seal.
In some embodiments, a desired decrease in temperature or downward ramp in temperature of the mixture in the reaction vessel 102 can be achieved via either passive cooling where the temperature difference between the reaction temperature and the ambient temperature causing cooling or active cooling, for example, where a Peltier cell is incorporated, and may be activated/deactivated in response to instructions from the controller 130.
In some embodiments, the memory (not shown) comprises instructions, which when executed by the processor(s) cause the controller 130 to cause stimulation of one or more fluorophore which may be provided within the reactive vessel(s) 102. For example, the controller 130 may communicate with the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate fluorophores.
Use of fluorescence as a detection signal can provide good sensitivity, and where the measurement components include multiple fluorescence detectors 128 configured to detect respective non-overlapping wavelength ranges so as not to interfere with one another, multiple channels of test and/or control reactions can be incorporated within a single reaction vessel 102. The measurement components can be configured to measure optical absorption, reflection, luminance output, and/or fluorescence.
Self-test capacity
In some embodiments, additional openings in the support 104 are provided to receive calibration or reference samples with specific optical characteristics. This can be used to self-calibrate or self-test the apparatus 100 during power up or measurement cycles by comparing the measured reference values against known values for the reference target.
Ultrasonic mixing
In some embodiments, the support 104 or heating block can be coupled to a vibrating mechanism or actuator such as an electromagnetic coil and slug. Actuation of this component can induce vibration in the reaction vessels mounted in the support. The excitation frequent can be in the range of Hz up to kHz. Where the excitation is above 20 kHz, it can be referred to as ultrasonic mixing. For high frequent or ultrasonic mixing, a piezoelectric actuator can be used. In some embodiments, the apparatus 100 includes an ultrasonic transducer configured so that the ultrasonic transducer can contact it through an opening in the support 104, with the ultrasonic transducer slightly lifting the reaction vessel so that it is not fully supported by the support 104. This may for allow efficient ultrasonic excitation of each reaction vessel only (i.e., without or with reduced excitation of the support itself) while otherwise allowing the reaction vessel 102 to be seated in good thermal contact at other rotation positions and associated measurement stations.
Barcode reading and image analysis
In some embodiments, the apparatus 100 includes a barcode reader 136, for example an RFID reader or image sensor. The support 104 may be configured to position a selected reaction vessel or associated disposable plastic assembly carrying or forming the vessel such that an attached label or feature is positioned in front of the barcode reader, RFID reader or image sensor. Where an image sensor is used, this can also be employed to confirm that the sample and reaction vessel or the disposable assembly that carries the reaction vessels have the correct reagents added, are assembled correctly, and are functional.
System for determining quantities of nucleic acid in a sample
Figure 14 is a block diagram of an example system 200 for quantifying one or more nucleic acids in a sample. For example, the sample may form part of a test mixture provided in a reaction vessel 102 of the apparatus 100. The test mixture may comprise the sample containing the one or more nucleic acids and a reagent mixture. In some embodiments, the test mixture further comprises at least one detection reagent, such as a fluorophore.
The system 200 comprises one or more processors 202 and memory 204. The processor(s) 202 may comprise one or more microprocessors, central processing units (CPUs), application specific instruction set processors (ASIPs), application specific integrated circuits (ASICs) or other processors capable of reading and executing instruction code. The memory 204 may comprise one or more volatile or non-volatile memory types. For example, the memory 204 may comprise one or more of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM) or flash memory. The memory 204 is configured to store program code accessible by the processor(s) 202. The program code comprises executable program code modules. In other words, memory 204 is configured to store executable code modules configured to be executable by the processor(s) 202. The executable code modules, when executed by the processor(s) 202 cause the system 200 to perform certain functionality, as described in more detail below.
Memory 204 comprises data 218 and program code 220. Data 218 comprises one or more of: first data set 222, second data set 224 and calibration data set 226. Program code 220 comprises one or more of: a temperature control module 206, an amplification detection point module 208, an amplification process start time determination module 210, and amplification duration determination module (or quantification module) 212.
It will be appreciated, however, that the program code or code modules of memory 204 which when executed by the processors 202 cause the system 202 to perform the methods of any one of Figures 15 to 18, may be stored instead in memory of the controller 130 of the apparatus 100, and the controller 120 may instead be configured to perform the functionality. In some embodiments, the methods of any one of Figures 15 to 18 may be performed by the system 200 and the controller 130 acting together, with some of the functionality being performed by the system 200 and some of the functionality being performed by the controller 130 of the apparatus 100.
The system 200 may comprises a network interface 214 and/or communications module 218 to facilitate communications with other systems, devices, and/or databases, for example, across a communications network (not shown), such as local area network(s) or wide area network(s), for example, the Internet. For example, the network interface 214 may facilitate communications between the system 200 and the controller 130 of the apparatus 100. The communications module 216 and/or network interface 214 may comprise a combination of network interface hardware and network interface software suitable for establishing, maintaining and facilitating communication over a relevant communication channel. The communications module 216 and/or network interface 214 may allow for wired or wireless communication between the system 200 and external devices, such as the controller 130 of the apparatus 100, and may use WiFi, USB, Bluetooth, or other communications protocols.
Process flows
Figure 15 is a process flow diagram of a method 1500 of quantifying one or more nucleic acids in a sample, according to one embodiment. The sample forms part of a test mixture comprising the sample, a reagent mixture and at least one detection reagent. The test mixture is provided within a reaction vessel 102 arranged to be received by the apparatus 100, for example, by support 104. The method 1500 may be implemented by the processor(s) 202 of system 200 executing instructions stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
At 1502, the test mixture is subjected to substantially isothermal conditions for a first period of time. For example, the temperature control module 206 of the controller 130 may send instructions to the heating element(s) 120 to cause the heating elements 120 to increase the temperature of the heater block 124 or support 104 and receive measurements from the temperature sensor(s) 122 to monitor the temperature of the test mixture until a set point temperature or within a range of a set point temperature (as stored in memory) which corresponds with substantially isothermal conditions is achieved. For example, the set point may be an initial isothermal temperature.
In some embodiments, responsive to the controller 130 detecting that the apparatus 100 has received the reaction vessel 102 (for example, the reaction vessel 102 is detected, for example, via a sensor, in the support 104), the controller 130 causes heating of the text mixture. However, it will be appreciated that various different actions may cause the controller 130 to heat the text mixture to the set point. Such actions may include, for example, detection by the controller 130 of a user activating a user- selectable activator to cause the amplification process to begin, detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus, or detection by the controller 130 of the vessel receiving a reaction liquid.
At 1504, the controller 130 records data indicative of the temperature of the test mixture over the first period of time. The first period of time spans from before the test mixture is heated to a time after the test mixture reaches an amplification temperature. In one example, the first period of time spans from a time when the test mixture is at an initial isothermal temperature for an initial isothermal time period. For example, the controller 130 may begin to record data indicative of the temperature once the controller 130 detects that the temperature of the test mixture is determined to be at the initial isothermal temperature.
The controller 130 records data indicative of the temperature of the test mixture from an initial isothermal temperature for an initial isothermal time period. The initial isothermal temperature may be between 58°C and 66°C, for example about 65°C. The first period of time may include the initial isothermal time period. The first period of time may be substantially the same duration as the initial isothermal time period. In one example, the first period of time may be between 1 and 2 minutes. For example, the initial isothermal time period may be between 1 and 2 minutes. The data indicative of the temperature of the test mixture over the first time period may be stored in a first data set. The controller 130 may store the first data set locally in memory and/or may communicate it to the system 200 to be stored in memory 204.
In some embodiments, the data indicative of the temperature of the test mixture over the first time period is temperature data. For example, the controller 130 may receive and record temperature data from the temperature sensor(s) 122. The temperature data may be time series data. The temperature data may comprise temperature measurements or readings, with each temperature measurement being associated with a time stamp indicative of the time at which the reading was obtained during the first period of time. The temperature readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
In some embodiments, the at least one detection reagent comprises one or more first fluorophores which vary with temperature. For example, the first fluorophore may be a decaying temperature dependent fluorescent dye, such as Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).. In these embodiments, the data indicative of the temperature may be first fluorescence data indicative of fluorescence emitted by the first fluorophore(s). The controller 130 may be configured to cause stimulation of the first fluorophore(s) and monitor the resulting fluorescence. For example, the controller 130 may transmit instructions to the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate the first fluorophore(s). The controller 130 monitors the emitted fluorescence by receiving first fluorescence data from the fluorescence detector(s) 128. The controller 130 records the first fluorescence data. The first fluorescence data may be time series data. The first fluorescence data may comprise fluorescence measurements or readings, with each fluorescence measurement being associated with a time stamp indicative of the time at which the reading was obtained during the first period of time. The fluorescence readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
The controller 130 may cause stimulation of the first fluorophore(s) in response to an action occurring. Such actions may include: (i) detection by the controller 130 of a user activating a user-selectable activator to cause the amplification process to begin; (ii) detection by the controller 130 of the vessel being placed in a vessel support of the apparatus; (iii) detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus; (iv) detection by the controller 130 of the vessel receiving a reaction liquid; or (v) detection by the controller 130 of heat being applied to the vessel or test mixture within the vessel. At 1506, once the test mixture has reached the amplification temperature, the controller 130 causes ramping of the temperature of the test mixture between a first temperature and a second temperature for a second or ramping period of time. The temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved.
The controller 130 (for example, the temperature control module 206) may be configured to ramp the temperature of the test mixture between the first and second temperatures by transmitting instructions to the heating elements 120, activating air flow and/or Peltier cell elements 116 to actively cool the test mixture, for example, by cooling the support 104, and monitoring the temperature based on readings from the temperature sensor(s) 122.
For example, the amplification temperature may be between 20 and 65 degrees Celsius. For example, the amplification temperature may be about 40 degrees Celsius or at about 65 degrees Celsius.
For example, the first temperature is ramped for a period of approximately 100 seconds and/or the second temperature is ramped for a period of approximately 90 seconds. For example, the ramping rate may be between 0.01 degrees Celsius per second and 5 degrees Celsius per second.
The first temperature is less than an amplification temperature effective to cause the amplification reaction, and the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature. For example, a temperature difference between the first and second temperatures may be between 2 and 40 degrees Celsius. For example, the first temperature may be about 25 degrees Celsius and the second temperature is about 40 degrees Celsius, and the isothermal temperature is about 40 degrees Celsius. For example, the first temperature may be about 50 degrees Celsius and the second temperature is about 65 degrees Celsius, and the isothermal temperature is about 65 degrees Celsius.
At 1508, the controller 130 records detection reagent data associated with the test mixture during a third period of time. The third time period may correspond with, or be a subset of, the second or ramping period of time.
At 1510, the amplification detection point module 208 determines one or more detection points based on detection reagent data recorded during the third period of time. Each detection point is indicative of sufficient amplification to allow for detection of a respective nucleic acid in the sample. The detection point may be indicative of the amplification time. The detection reagent(s) may be at least one second fluorophores where fluorescence emitted by the second fluorophore(s) varies with nucleic acid binding. The detection reagent data may comprise fluorescence data indicative of fluorescence emitted by the second fluorophore(s) during the second period of time. The second fluorophore(s) may be the first fluorophore(s); that is the first fluorophore(s) used in the above embodiment (where data indicative of the of the temperature is fluorescence data indicative of fluorescence emitted by the first fluorophore(s)) is again used as the second fluorophore(s). The second fluorophore(s) may be separate fluorophore(s) to the first fluorophore(s) and may be the same type of fluorophore or a different type of fluorophore. For example, the second fluorophore may be a decaying temperature dependent fluorescent dye, such as Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
The controller 130 may be configured to cause stimulation of the second fluorophore(s) and to monitor the resulting fluorescence over the third time period. For example, the controller 130 may transmit instructions to the light source 126 to cause the light source 126 to emit light at one or more select frequencies to selectively stimulate the second fluorophore(s). The controller 130 monitors the emitted fluorescence by receiving second fluorescence data from the fluorescence detector(s) 128. The controller 130 records the second fluorescence data. The second fluorescence data may be time series data. The second fluorescence data may comprise fluorescence measurements or readings, with each fluorescence measurement being associated with a time stamp indicative of the time at which the reading was obtained during the third period of time. The fluorescence readings may be discrete, obtained at regular or irregular interval time periods, or may be continuous.
The controller 130 may cause stimulation of the second fluorophore(s) in response to an action occurring. Such actions may include: (i) detection by the controller 130 of a user activating a user-selectable activator to cause the amplification process to begin; (ii) detection by the controller 130 of the vessel being placed in a vessel support of the apparatus; (iii) detection by the controller 130 of a lid being coupled to the vessel placed in the vessel support of the apparatus; (iv) detection by the controller 130 of the vessel receiving a reaction liquid; (v) detection by the controller 130 of heat being applied to the vessel or test mixture within the vessel; (vi) detection by the controller 130 of the test mixture being at a predetermined temperature, such as the amplification temperature; or (vii) determination that an initial isothermal time period has passed.
In some embodiments, the memory of the apparatus 100 comprises the amplification detection point module 208 and the processor of the apparatus 100 executes the module 208 to cause the controller 130 to determine the amplification detection point(s).
In some embodiments, the amplification detection point module 208 is stored remote from the apparatus 100, for example in memory 204 of system 200. In such embodiments, the data indicative of fluorescence emitted by the second fluorophore(s) during the ramping period of time is provided to the system 200. For example, the controller 130 may be configured to transmit the data to the system 200. The data may be transmitted as the second data set once all readings have been obtained, or the data may be streamed as it is being received by the controller 130 or transmitted in chunks as the chunks are collated, with each chunk comprising a predetermined number of readings.
At 1512, the amplification process start time determination module 210 determines a start time of the amplification process based on the first data indicative of the temperature of the test mixture over the first time period.
In some embodiments, the memory of the apparatus 100 comprises the amplification process start time determination module 210 and the processor of the apparatus 100 executes the module 210 to cause the controller 130 to determine the start time of the amplification process.
In some embodiments, the amplification process start time determination module 210 is stored remote from the apparatus 100, for example in memory 204 of system 200. In such embodiments, the data indicative of the temperature of the test mixture over the first time period set is provided to the system 200. For example, the controller 130 may be configured to transmit the data to the system 200. The data may be transmitted as the first data set once all readings have been obtained, or the data may be streamed as it is being received by the controller 130 or transmitted in chunks as the chunks are collated, with each chunk comprising a predetermined number of readings.
One method 1600 of determining the amplification process start time is described in more detail below with reference to Figure 16.
At 1514, the amplification duration determination module 212 determines the amplification duration for the at least one or more nucleic acids in the sample based on the detection point and the amplification start time. The amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
In some embodiments, the controller 130 and/or system 200 provides the determined amplification duration(s) for respective nucleic acid(s) in the sample to the user interface 132 or display of the apparatus 100 for presentation to a user. In some embodiments, the memory of the apparatus 100 comprises the amplification duration determination module 212 and the processor of the apparatus 100 executes the module 212 to cause the controller 130 to determine the amplification duration. If the amplification process start time determination module 210 is not executing locally on the apparatus, the system 200 transmits the amplification process start time to the controller 130.
In some embodiments, the amplification duration determination module 212 is stored remote from the apparatus 100, for example in memory 204 of system 200. In such embodiments, the amplification start time is provided to the system 200, if the amplification process start time determination module 210 is not executing locally on the system 200. In such embodiments, the determined detection point(s) of nucleic acid(s) in the sample are provided to the system 200, if the amplification detection point module 208 is not executing locally on the system 200. For example, the controller 130 may be configured to transmit the determined amplification start time and/or detection point(s) to the system 200.
One method 1700 of determining the amplification duration is described in more detail below with reference to Figure 17.
It will be appreciated that in some embodiments of method 1500, step 1504 is omitted, and at 1514, the amplification duration determination module 212 determines the amplification duration for the at least one or more nucleic acids in the sample based on the detection point. In other words, there is no determination of amplification process start time as discussed in more detail in Figure 16.
It will also be appreciated that in some embodiments of method 1500, step 1506 is omitted (i.e., there is no ramping between two temperatures), and instead the amplification temperature is maintained.
Figure 16 is a process flow diagram of a method 1600 of determining an amplification process start time, according to some embodiments. The method 1600 may be implemented by the processor(s) 202 of system 200 executing instructions (such as the amplification process start time determination module 210) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification process start time determination module 210) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
At 1602, the amplification process start time determination module 210 determines data indicative of the temperature of the test mixture over the first time period. The data may comprise the data recorded by the controller 130 at 1504 of method 1500 of process flow diagram Figure 15. In some embodiments, the amplification process start time determination module 210 is stored and executed at system 200 and the system 200 is configured to receive the data from the controller 130, as a first data set, in streamed format, or in chunks of data, as described above. It will be appreciated that method 1600 can execute while data is still being recorded and/or transmitted to the amplification process start time determination module 210. In some embodiments the data is temperature data and in some embodiments the data is fluorescence data.
In some embodiments, the amplification process start time determination module 210 determines whether a threshold data acquisition time has elapsed. The threshold data acquisition time may be a period of time considered sufficient for the test mixture to be at temperature and stable.
At 1604, the amplification process start time determination module 210 determines a start time of the amplification process as being a time at which a start time detection condition is met.
In one example, the amplification process start time determination module 210 determines the start time as being a time at which the temperature of the test mixture is within a threshold range of the amplification temperature.
In one example, the amplification process start time determination module 210 determines the start time as being a time at which a fluorescence level of the of the first fluorophore of the first fluorophore data meets a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature.
In one example, the amplification process start time determination module 210 determines the start time as being a time at which there is a maximum rate of change of gradient of the first fluorophore of the first fluorophore data.
In one example, the amplification process start time determination module 210 determines the start time as being a time at which a steady state emission level of fluorescence of the first fluorophore is achieved and is indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids.
In some embodiments, the amplification process start time determination module 210 plots a graph of fluorescence against time using the third data set and determines the start time from the plotted graph.
At 1606, the amplification process start time determination module 210 provides the determined start time to the amplification duration determination module 212.
Figure 17 is a process flow diagram of a method 1700 of determining one or more detection points indicative of sufficient amplification to allow for detection of one or more respective nucleic acids in the sample, according to some embodiments. The method 1700 may be implemented by the processor(s) 202 of system 200 executing instructions (such as the amplification detection point module 208) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification detection point module 208) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another.
At 1702, the amplification detection point module 208 determines second fluorescence data indicative of fluorescence emitted by the second fluorophore(s) during the second or ramping period of time as captured while the test mixture was being ramped between a first temperature and a second temperature at 1506 of method 1500, as described above.
In some embodiments, the amplification detection point module 208 is stored and executed at system 200 and the system 200 is configured to receive the second fluorescence data from the controller 130, as a second data set, in streamed format, or in chunks of data, as described above. It will be appreciated that method 1700 can execute while data is still being recorded and/or transmitted to amplification detection point module 208.
In some embodiments, the amplification detection point module 208 interpolates and/or extrapolates the second fluorescence data.
At 1704, the amplification detection point module 208 determines a background fluorescence level for the assay before significant amplification occurs.
At 1706, responsive to determining that a fluorescence level of the second fluorophore of the fluorescence data meets an amplification detection condition, determining by the amplification detection point module 208 a time at which the amplification detection condition was met as a detection point indicative of sufficient amplification to allow for detection of respective nucleic acids in the sample.
At 1708, the amplification detection point module 208 provides each determined detection point(s) to the amplification duration determination module 212.
Figure 18 is a process flow diagram of a method 1800 of quantifying one or more nucleic acids in the sample, according to some embodiments. The method 1800 may be implemented by the processor(s) 202 of system 200 executing instructions (such as amplification duration determination module 212) stored in memory 204, or by the processor(s) of the controller 130 of the apparatus 100 executing instructions (such as the amplification duration determination module 212) stored in memory (not shown) or by both the system 200 and controller 130 cooperating with one another. At 1802, the amplification duration determination module 212 determines the determined detection point(s) and the determined start time.
At 1804, the amplification duration determination module 212 determines an amplification duration for each of the nucleic acid(s) based on respective determined detection point(s) and the start time. The amplification duration is indicative of the quantity of the one or more nucleic acids in the sample. In some embodiments, the amplification duration for each of the nucleic acid(s) is calculated as the difference between the respective detection point(s) and the determined start time.
At 1806, the amplification duration determination module 212 determines a suitable calibration dataset of known concentrations. For example, the calibration dataset may have been determined using the same quantification time determination methods as described in relation to method 1700. This calibration dataset may be saved from calibration tests run previously or run in separate tubes in the same instrument at the same time at the test in progress. The calibration dataset may be stored locally to the amplification duration determination module 212, or may be retrieved from a remote database, for example.
At 1808, the amplification duration determination module 212 determines the quantification for each nucleic acid based on the respective determined amplification duration and the calibration dataset. This amplification duration determination module 212 takes into account the dynamics of the specific isothermal amplification method (whether under ramping or not) and which may be linear, exponential or some other equation or a curve fit from experimental data.
In some embodiments, the amplification duration determination module 212 provides the determined quantification for respective nucleic acid(s) in the sample to the user interface 132 or display of the apparatus 100 or system 200 for presentation to a user.
As an example, assume the amplification duration determination module 212 determines an amplification duration or “Test Time” of 700 seconds, the assay type is linear in its response, and the following assay calibration data is available:
800 viral copies per mL has and test time of 400 seconds
600 viral copies per mL has and test time of 600. seconds
400 viral copies per mL has and test time of 800 seconds
200 viral copies per mL has and test time of 1000 seconds.
The amplification duration determination module 212 will interpolate the assay calibration data between the 600 second value and 800 second values, and determine a quantification of 500 viral copies per mL. Applications
Applications of the apparatus and methods described herein include diagnostic testing, particularly relating to a compact portable test instrument suitable for use in medical diagnostic at the Point-of-Care (POC) and in Physician’s Office Laboratories (POL).
The described embodiments of the present invention include nucleic acid amplification and detection apparatus that are configured to receive only one or two measurement tubes contained within a single consumable assembly and are therefore suitable for portable, point of care, or other field applications. The described heated support/rotor arrangements enable multiple measurements, self-calibration and mixing functions to be performed with respect to a small number of reaction vessels or test tubes that are contained within in or are part of a disposable cartridge or vessel assembly. These features allow reduced complexity and make possible a compact, portable and relative low cost apparatus.
Notwithstanding the above, it will be apparent that in other embodiments a nucleic acid amplification and detection apparatus can in general be configured to receive any practical number of reaction vessels.
Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.
The present disclosure includes the following non-limiting Examples.
EXAMPLES
Example 1: Exemplary optimal and non-optimal amplification temperatures
To highlight the optimal temperature, Figure 1A depicts an example rate of amplification versus temperature curve for a hypothetical amplification configuration. The optimal temperature, i.e. the temperature at which amplification occurs most rapidly, is labelled tempo. Other example temperatures, tempa, tempb and tempc, which are lower than tempo, are also labelled. The highlighted section indicates the range of temperatures at which amplification will occur based on variation in reaction conditions, and for temperatures above and below this range, for example tempa, amplification will either not occur (tempp), or will not be detectable (tempa). This temperature range may only be a couple of degrees or could be as much as degrees, for example, between 60-70 degrees Celsius. Small changes in the amplification liquid temperature may result in a large range in the rate at which isothermal amplification will occur. Figure IB provides representations of isothermal amplification reference curves of the amplification configuration of Figure 1A, produced with amplification liquid temperature at tempa, tempt,, tempc, and tempo. As tempa is outside the amplification temperature range, no detectable amplification occurs. Amplification occurs more rapidly where the reaction temperature is tempt,, and more rapidly again at tempc. The most rapid amplification occurs where the temperature of the amplification fluid is at the optimal temperature, tempo. Significant changes in amplification will affect the accuracy of the reference curve-based quantification.
Figure 2 depicts the block and amplification liquid temperatures versus time for an existing method of quantitative isothermal amplification when a predetermined non- optimal temperature is used for performing quantitative isothermal amplification. The temperature for the reaction is selected to be at the at the lower end of the temperature range at which amplification will occur. Both the block temperature and the amplification liquid temperature are depicted, and both increase (or ramp up) to the selected non- optimal temperature, which is maintained as indicated by the horizonal portions of the block temperature and amplification liquid curves. For example, if the optimal temperature of the reaction is 55 degrees Celsius and during the sample run the instrument is running at 58 degrees Celsius, the reaction is not running at the optimal temperature, and this will result in a different quantification when compared to standards (which are generally not run on the same day). It will be apparent to the skilled person that running standards at the sub-optimal temperature is expensive and requires increased time for testing.
Example 2: Exemplary nucleic acid amplification with ramping
Figure 3 provides an exemplary schematic of block temperature ramping. For example, tempo indicates an optimal temperature for isothermal amplification, and the range of temperatures at which amplification occur is shaded and labelled ‘Amplification temperature range’ . The block temperature is ramped up and down between tempA and temps (as indicated by the triangle wave labelled ‘Block temperature’). The area shaded within Figure 3A and 3B indicates the optimal temperature for the reaction and corresponds to rate of change of amplification as it corresponds to temperature (see e.g., Figure 1A).
Example 4: Exemplary methods of detecting and quantifying nucleic acids
An exemplary schematic of the detection point of the one or more nucleic acids is depicted in Figure 4 which denotes the cross point (Cp) or detection point over a number of cycles. The detection point or Cp will be reached as the amplification produces sufficient fluorescence above the background signal.
Figure 5A depicts the values for the different standard curves over time performed by isothermal amplification. The standard curves represent different starting concentrations of template nucleic acids. The earlier the Cp, the higher the initial nucleic acid template within the sample. The Cp of the standard curves can be alternatively plotted based on quantification time (Tq), where a sample with a lower Cp will have a lower Tq (e.g. CpA and TqA) and accordingly higher template nucleic acids, whereas a sample with a higher Cp will have a higher Tq (e.g. CpE and TqE) and accordingly low template nucleic acids. The quantification time (Tq) can represent the total time that the amplification liquid is within the correct range.
Figure 5B depicts the Cp of the standard curves plotted against the known template concentrations of the standards. A sample can be cross-referenced to standards and the target nucleic acid concentration calculated (CqX).
Example 6: Standard amplification at constant temperature and calculation of start time
Loop-mediated amplification (LAMP) was used to assess isothermal nucleic acid amplification and determination of a constant start time for quantification. Sample reactions are shown in Table 1 below. Samples were run at increasing concentrations at 65 degrees Celsius optimum running temperature in a final volume of lOOpL on an Axxin T8 instrument with beads.
Table 1: Quantification dilutions
As shown in Figure 10, the amplification curve rise times are bunched close together or even overlay and do not provide good quantification separation. The fluorescence fluorophore HEX used in this test has fluorescence that falls with increasing temperature (decay). This test demonstrated the typical florescence curve right at the start of the test where it falls to steady state. This curve shape can be used to determine a constant start time, (tO) for the assay and quantification calculation. The shaded region is the starting region of the curve.
The graph in Figure 11 shows an expanded view of the shaded starting region of the curve in Figure 10. Two example algorithm methods to determine a consistent quantification starting time for the quantification calculation by interpreting the fluorescence warm up curve shape were then determined. As shown in Figure 11A, the first method used the crossing point (A) where the assay temperature crossed a defined level of fluorescence difference corresponding to temperature difference as the assay warms up approaching the running temperature (amplification temperature range). The crossing point is then used to determine the start time. The line at a fixed level of fluorescence (B) corresponds to a temperature difference from the stable running temperature. The threshold is used as the estimate of the start time using post-processing. The threshold is a certain amount of time before the stable fluorescence. As shown in Figure 11B, the second method utilises the inflection point or point of maximum rate of change of gradient (C) in fluorescence temperature response to define the starting time point (using post-processed data) as the assay is approaching the running temperature (amplification temperature range).
Example 2: Amplification with temperature ramping
To demonstrate the use of ramping to spread out and provide enhanced separation for the calculation and determination of quantification, temperature cycling with 100 second ramp down to 50 degrees Celsius followed by 90 seconds at 65 degrees Celsius was performed using reagents described in Table 1.
The individual amplification time points were determined by several methods. As shown in Figure 12 the point at which the assay exponential curve crossed over a line that extrapolated a constant level above the number of amplifications thermal ramping curve for the assay response in the test was determined. The level increased slowly over time due to a low level of non-specific amplification and/or other effects which increased the background fluorescence of the assay curve.
In this example, tO was the starting time point and time points tl, t2, t3, t4 etc corresponded to specific nucleic acid amplification timepoints. The quantification calculation algorithm takes into account the time difference, for example t2 minus tO compared to either 1) known previously determined quantification curves, or 2) known quantification samples run at the same time. The quantification algorithm was also tested using the number of ramp cycles including the proportion of the last cycle as an alternative or in combination with the assay time. For example, the 102 sample has time of tO minus t2 and has a ramp cycle count of approximately 6.8 cycles with 6 full cycles and some proportion of a cycle at the start and in the last partial cycle when exponential amplification response occurs. This was calculated from analysis of all datapoints in the measured dataset.
Example 3: LAMP with initial isothermal period followed by temperature ramping cycles
A test workflow was performed using LAMP with an initial isothermal period of 50 degrees Celsius for 15 minutes followed by a series of temperature ramping cycles of 100 seconds at 50 degrees Celsius followed by 90 seconds at 65 degrees Celsius (Figure 13). This provided the following advantages:
• A steady temperature at the start of the test allowed a clear determination of test start time using the methods described above.
• The use of ramp cycling spread out the quantitative results once the assay approached the exponential amplification region of the assay.

Claims

1. A method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) subjecting the test mixture to substantially isothermal conditions for a first period of time, wherein the first period of time spans a time from before the test mixture is heated to an amplification temperature to a time after the test mixture reaches the amplification temperature;
(ii) recording, over the first time period, data indicative of the temperature of the test mixture;
(iii) determining, based on a first data set comprising the data indicative of the temperature of the test mixture over the first time period, a start time of an amplification process as being a time at which a start time detection condition is met;
(iv) ramping the temperature of the test mixture between a first temperature and a second temperature for a second period of time once the test mixture has reached the amplification temperature, wherein the temperature is ramped until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature;
(v) recording, over a third period of time, detection reagent data associated with the test mixture, wherein the third period of time is at least a subset of the second period of time;
(vi) determining at least one detection point based on a second data set comprising the detection reagent data, wherein the at least one detection point is indicative of sufficient amplification of a respective at least one nucleic acids of the one or more nucleic acids in the sample; and
(vii) determining an amplification duration for the at least one nucleic acid in the sample based on the respective at least one detection point and the amplification start time, wherein the amplification duration is indicative of the quantity of the respect at least one nucleic acid of the one or more nucleic acids in the sample.
2. A method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining a first data set comprising data indicative of the temperature of the test mixture over a first time period, wherein the first time period spans a time from before the test mixture was heated to an amplification temperature to a time after the test mixture reached the amplification temperature;
(ii) determining based on the first data set, a start time of an amplification process as being a time at which a start time detection condition is met;
(iii) determining a second data set comprising detection reagent data associated with the test mixture and recorded during a third period of time, the third period of time being at least a subset of a second period of time during which and once the test mixture had reached the amplification temperature, the temperature of the test mixture was ramped between a first temperature and a second temperature until amplification of the at least one nucleic acid of the one or more nucleic acids in the sample was achieved, wherein the first temperature was less than an amplification temperature effective to cause the amplification reaction, and wherein the second temperature was within or exceeded an amplification temperature range comprising the amplification temperature;
(iv) determining, based on the second data set, at least one detection point indicative of sufficient amplification of a respective at least one nucleic acid of the one or more nucleic acid in the sample; and
(v) determining an amplification duration for the at least one nucleic acid in the sample based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the one or more nucleic acids in the sample.
3. A method of quantifying one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) monitoring a temperature of the text mixture over a first period of time;
(ii) after the temperature of the text mixture reaches the amplification temperature, ramping, at a ramping rate, the temperature of the test mixture between a first temperature and a second temperature for a second period of time, until amplification of at least one nucleic acid of the one or more nucleic acids in the sample is achieved, wherein the ramping rate is between 0.01 degrees Celsius per second and 5 degrees Celsius per second, wherein the first temperature is less than an amplification temperature effective to cause the amplification reaction, and the second temperature is within or exceeds an amplification temperature range comprising the amplification temperature, and wherein a temperature difference between the first and second temperatures is between 2 and 40 degrees Celsius;
(ii) recording, over a third time period, detection reagent data associated with the test mixture, wherein the third time period is at least a subset of the second period;
(iii) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(v) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
4. A method for determining an initial quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining detection reagent data associated with the test mixture, the detection reagent data having been recorded during a third period of time once the test mixture had reached the amplification temperature, and while the temperature of the test mixture was ramped between a first temperature and a second temperature until amplification of the at least one nucleic acid of the one or more nucleic acids in the sample was achieved, wherein the ramping rate was between 0.01 degrees Celsius per second and 5 degrees Celsius per second, wherein the first temperature was less than an amplification temperature effective to cause the amplification reaction and the second temperature was within or exceeded an amplification temperature range comprising the amplification temperature, and wherein a temperature difference between the first and second temperatures is between 2 and 40 degrees Celsius;
(ii) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(iii) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
5. The method of any one of the preceding claims, wherein the at least one detection reagent is at least one first fluorophore, wherein fluorescence emitted by the first fluorophore of the at least one fluorophore varies with nucleic acid binding, and wherein the detection reagent data comprises fluorescence emitted by the at least one first fluorophore of the test mixture.
6. The method of any one of the preceding claims, wherein the at least one fluorophore is at least one decaying temperature dependent fluorescent dye.
7. The method of claim 6, wherein the decaying temperature dependent fluorescent dye is Hexachloro-fluorescein (HEX), Rhodamine B (RhB), 5’ 6-fluorescein (FAM) or carboxyrhodamine (ROX).
8. The method of claim 1, wherein subjecting the test mixture to substantially isothermal conditions comprises heating the test mixture to a temperature of between 20 and 65 degrees Celsius.
9. The method of claim 8, wherein the temperature is at about 40 degrees Celsius or at about 65 degrees Celsius.
10. The method of any one of claims 1, or 5 to 9 when dependent directly or indirectly on claim 1, wherein: (i) the substantially isothermal condition is at a temperature of about 40 degrees Celsius, and during ramping the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius; or
(ii)the substantially isothermal condition is at a temperature of about 65 degrees Celsius, and during ramping the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
11. The method of any one of claims 1, 2 or 5 to 10 when dependent directly or indirectly on claim 1 or 2, wherein a temperature difference between the first temperature and the second temperature is between 2 and 40 degrees Celsius.
12. The method of any one of claims 1, 2 or 5 to 11 when dependent directly or indirectly on claim 1 or 2, wherein during ramping:
(i) the first temperature is about 25 degrees Celsius and the second temperature is about 40 degrees Celsius; or
(ii)the first temperature is about 50 degrees Celsius and the second temperature is about 65 degrees Celsius.
13. The method of any one of claims 1, 2 or 5 to 12 when dependent directly or indirectly on claim 1 or 2, wherein the temperature is ramped at a ramping rate of between 0.01 degrees Celsius per second and 5 degrees Celsius per second.
14. The method of any one of claims 1, 2 or 5 to 12, wherein during ramping the first temperature is ramped for a period of approximately 100 seconds and/or the second temperature is ramped for a period of approximately 90 seconds.
15. The method of claim 1 or 2 or any one of claims 5 to 14 when dependent on claim 1 or claim 2, wherein recording, over the first time period, data indicative of the temperature of the test mixture comprises receiving temperature measurements from a sensor of the apparatus configured to sense, directly or indirectly, a temperature of the test mixture.
16. The method of claim 15, wherein the start time detection condition is a threshold range of the amplification temperature.
17. The method of claim 1 or 2 or any one of claims 5 to 14 when dependent on claim 1 or claim 2, wherein the test mixture comprises at least one at least one second fluorophore that varies with temperature, and wherein recording, over the first time period, data indicative of the temperature of the test mixture comprises determining fluorescence emitted by the at least one second fluorophore over the first time period.
18. The method of claim 17 when dependent directly or indirectly on claim 5 , wherein the at least one second fluorophore is the at least one first fluorophore.
19. The method of claim 17 or claim 18, wherein the start time detection condition is a fluorescence level threshold, wherein the fluorescence level threshold is greater than or equal to a steady state fluorescence emission level of the test mixture at the amplification temperature.
20. The method of claim 17 or claim 18, wherein the start time detection condition is a maximum rate of change of gradient of the at least one second fluorophore.
21. The method of claim 17 or claim 18, wherein the start time detection condition is a steady state emission level of fluorescence, the steady state emission level being indicative of the test mixture approaching the amplification temperature required to initiate amplification of the of one or more nucleic acids.
22. The method of any one of the preceding claims, further comprising calculating the quantity of the at least one nucleic acid based on the respective amplification duration.
23. The method of any one of the preceding claims, wherein the amplification duration is the time difference between the detection point and the start time.
24. The method of any one of claims 1 to 23, wherein the amplification duration is a number of ramping cycles.
25. The method of any one of the preceding claims, further comprising comparing the amplification time duration to standards indicative of known amplification time durations for known concentrations of nucleic acids to calculate the quantity of the at least one nucleic acid.
26. The method of any one of the preceding claims, wherein the test mixture further comprises a DNA polymerase with strand-displacement activity.
27. The method of claim 26, wherein the DNA polymerase is selected from the group consisting of phi29 or Bsu large fragment and Bst.
28. The method of any one of the preceding claims, wherein the test mixture further comprises an additive to lower the melting temperature (Tm) of the one or more nucleic acids in the sample.
29. The method of claim 27, wherein the additive is betaine.
30. The method of any one of the preceding claims, wherein the one or more nucleic acids is DNA or RNA.
31. The method of claim 30, wherein the one or more nucleic acids is RNA and the test mixture further comprises a reverse transcriptase.
32. A method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture, wherein the test mixture comprises (i) at least one detection reagent, (ii) a reagent mixture, and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) subjecting the test mixture to substantially isothermal conditions for a first period of time, wherein the first period of time spans a time from before the test mixture is heated to an amplification temperature to a time after the test mixture reaches the amplification temperature;
(ii) recording, over the first period of time, data indicative of the temperature of the test mixture;
(iii)determining, based on a first data set comprising the data indicative of the temperature of the test mixture over the first time period, a start time of an amplification process as being a time at which a start time detection condition is met;
(iv) recording detection reagent data associated with the test mixture over a third period of time when the test mixture had reached the amplification temperature;
(v) determining, based on a second data set comprising the detection reagent data, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(vi) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
33. A method of determining a quantity of one or more nucleic acids in a sample forming part of a test mixture provided in a vessel of an apparatus, wherein the test mixture comprises: (i) at least one detection reagent; (ii) a reagent mixture; and (iii) the sample containing the one or more nucleic acids, the method comprising:
(i) determining a first data set comprising data indicative of the temperature of the test mixture over a first time period, wherein the first time period spans a time from before the test mixture was heated to an amplification temperature to a time after the test mixture reached the amplification temperature;
(ii) determining based on the first data set, a start time of the amplification process as being a time at which a start time detection condition is met;
(iii) determining a second data set comprising detection reagent data associated with the test mixture, wherein the detection reagent data was recorded during a third time period when the test mixture had reached the amplification temperature;
(iv) determining, based on the second data set, at least one detection point indicative of sufficient amplification to allow for detection of a respective at least one nucleic acid of the one or more nucleic acids in the sample; and
(v) determining an amplification duration for the at least one nucleic acid based on the respective at least one detection point and the start time, wherein the amplification duration is indicative of the quantity of the at least one nucleic acid of the one or more nucleic acids in the sample.
34. A system comprising: one or more processors; and memory comprising computer executable instructions, which when executed by the one or more processors, cause the system to perform the method of any one of claims 1 to 33.
35. A computer-readable storage medium storing instructions that, when executed by a computer, cause the computer to perform the method of any one of claims 1 to 33.
36. The computer-readable storage medium of claim 35, wherein the computer- readable storage medium is a non-transient computer-readable storage medium.
PCT/AU2025/050174 2024-02-27 2025-02-27 Quantitative nucleic acid amplification and detection apparatus and methods thereof Pending WO2025179343A1 (en)

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