HK1195340B - Oscillating amplification reaction for nucleic acids - Google Patents

Abstract

One embodiment of the present invention provides for a method for amplifying a template of nucleic acid target sequence contained in a sample. The method includes contacting the sample with an amplification reaction mixture containing a primer complementary to the template of nucleic acid target sequence. A temperature of the reaction is oscillated between an upper temperature and a lower temperature wherein the change in temperature is no greater than about 20℃ during a plurality of temperature cycles. The template of nucleic acid target sequence is amplified.

Description

Oscillating amplification reaction for nucleic acids

Cross reference to related applications

This application claims priority and benefit from U.S. provisional patent application No. 61/477,437 entitled "oscillatory amplification reaction for nucleic acids" filed on 20/4/2011, and the specification and claims of which are incorporated herein by reference.

This application claims priority and benefit of U.S. provisional patent application No. 61/477,357 entitled "integrated apparatus for nucleic acid detection and identification" filed on 20/4/2011, and the specification and claims of which are incorporated herein by reference.

Declaration of federally sponsored research or development

Not applicable.

Incorporation by reference of material submitted on optical discs

Not applicable.

Copyrighted material

Not applicable.

Concerning sequence tables, or computer programs

Applicants submit a sequence listing herein that is a text file, entitled 041812_ ST25.txt, created at 4 months and 20 days 2012, having 10K kilobytes, conforming to ASCII, and incorporated herein by reference.

Background

Field of the invention (technical field)：

Embodiments of the invention relate to methods and apparatus for template-dependent amplification of nucleic acid target sequences by oscillating the reaction temperature over a small range, preferably over a range of no greater than 20 ℃, during any given thermal polymerization cycle.

Background art:

note that the following discussion refers to a number of publications and documents. The discussion of these documents is given herein for a more complete background of the scientific principles and is not to be taken as an admission that these publications are prior art for patentability determination.

Amplification of nucleic acids is one of the most indispensable techniques in molecular biology, and is widely used in research, genetic testing, agriculture, and forensic medicine. The most common amplification method is the Polymerase Chain Reaction (PCR), in which the dominance of a specific nucleic acid target sequence increases exponentially in solution (see U.S. Pat. nos. 4,683,195, 4,683,202, and 4,800,159). The PCR reaction employs two oligonucleotide primers that hybridize to opposite strands of the DNA duplex upstream (5 ') or downstream (3') of the target sequence to be amplified. A (usually thermostable) DNA polymerase is used to extend the hybridized primers in the 5 '→ 3' direction by the addition of deoxynucleoside triphosphates (dNTPs) in order to "copy" the target sequence and produce a double stranded DNA product. By cycling the temperature of the reaction mixture (typically 95 ℃ C.), the two strands of DNA can be separated at high temperature, which allows them to act as templates for further primer binding and polymerization at lower temperatures (e.g., 55 ℃ and 60 ℃ C.). After repeating this process multiple times, a single target sequence can be amplified into billions of copies.

Although PCR is a well-equipped, gold-labeled amplification method in a laboratory, it is quite complex, requires expensive and complex thermal cycling equipment with active heating and cooling heating elements and precise temperature control, and requires a trained technician to collect meaningful results. For example, most PCR reactions require rapid and precise cycling between at least two temperatures (e.g., 95 degrees and 57 degrees), which typically results in the use of expensive and inefficient Peltier motors (thermoelectric cooling mechanisms) and precise temperature control elements. Such inherent limitations make PCR unsuitable for the development of cost-effective, point-of-care nucleic acid diagnostics, which is useful in the absence of supporting laboratory infrastructure. To obviate the large resource requirements of some PCRs, various "isothermal" amplification techniques have been developed over the past few decades. In such reactions, nucleic acids can be amplified at a single temperature, eliminating the need for expensive thermal cyclers, and making them more suitable for use in low cost diagnostic equipment. Examples include Nucleic Acid Sequence Based Amplification (NASBA), helicase mediated amplification, strand displacement amplification, loop mediated isothermal amplification (LAMP), and the like. However, these isothermal amplification methods typically require 60-90 minutes amplification time (due to slow in vitro kinetic enzymatic processes) and precise temperature control at a single temperature point to accommodate extremely stringent amplification reactions, again lacking the robustness and speed required for in-situ diagnostic applications.

Template-dependent nucleic acid amplification is the basis for nucleic acid-based molecular diagnostics. Robust, low cost, rapid, point-of-care nucleic acid diagnostics are urgently needed in health care, agriculture, and in the context of the biological arts and warfare. However, assay chemistry strategies associated with existing PCR or isothermal amplification have significant engineering and robustness limitations, which make such amplification methods expensive and impractical for resource-limited situations (where nucleic acid-based molecules can maximally impact emergency disease prevention and control). Considerable improvements in nucleic acid amplification must be made to provide an affordable and robust diagnosis in the absence of specialized laboratory infrastructure.

Conventional PCR relies on high specificity and rapid thermal cycling, typically with temperature variations of up to 40 ℃. Such amplification methods require expensive instruments to rapidly heat and (especially) cool the PCR reaction mixture, and in addition precisely maintain the solution temperature. Isothermal nucleic acid amplification methods, while not requiring complex thermal cycling equipment, are generally slow (at least 60 minutes of reaction time), unreliable, and require precise temperature calibration.

In PCR thermocycling methods, the PCR cycler must have good temperature control to maintain temperature uniformity within the sample and a typical sample heating (and/or cooling) rate of at least 2 ℃/sec. Temperature control is typically achieved by a feedback loop system, while temperature uniformity is achieved by a highly thermally conductive but bulky material such as copper. High heating rates are achieved by implementing a Proportional Integral Derivative (PID) control method, which is limited by maximum dissipated power and heat capacity. High cooling rates are rather difficult to achieve and large volume systems require forced cooling by means of thermoelectric elements (p.wilding, m.a.shoffner and l.j.kricka, clin.chem., 1994, 40, 1815-. These PCR machines are complex and power consuming devices. Because the system is bulky, its thermal time constant is in minutes rather than seconds, which results in long transition times and unwanted PCR byproducts. The high power consumption makes it impossible to prepare a battery-powered and portable PCR system.

With the recent development of silicon technology-based micromachining and biological micro-electromechanical systems (bioMEMS), many groups of subjects worldwide have begun to develop microPCR (μ PCR), which is a central part of the lab-on-a-chip (1ab-on-a-chip) or micro total analysis system (μ TAS). Researchers follow two basic approaches: a stationary system that cycles temperatures, a flow system with three zones at different temperatures. Both of these systems have their advantages and disadvantages. The immobilization system cycles the temperature of the chamber in order to change the temperature of the PCR solution. It does not require a pumping system or other means for moving the PCR sample back and forth. Flow-through systems typically have three zones at constant temperature. The sample changes temperature by merely moving between zones of different temperatures. This PCR system is faster than the first, but it requires an execution mechanism to move the sample back and forth. In both cases, the heater is integrated with the PCR system, so it is uneconomical to discard the device to avoid cross-contamination after only a single test is performed. The two forms exhibit major advantages: the cycle time is reduced compared to conventional devices, and the sample volume used is reduced. However, these PCR chips use a substrate material such as silicon that requires an expensive and complicated manufacturing process, resulting in a very high unit price. Furthermore, due to the extremely small reaction volume (< μ l) required to obtain an improved surface/volume ratio and the type of material used for μ PCR chips, some effects not much the same as conventional PCR become apparent, including nonspecific adsorption of biological samples, inhibition, sample evaporation, and bubble formation. Other current efforts are also directed to the development of temperature cycling reaction microchips that integrate fixed chamber and continuous flow PCR to perform efficient temperature cycling of flow-through microchannel PCR chips, while having the flexibility to vary the number of cycles and the number of temperature zones in the fixed chamber in the PCR chip. However, the effectiveness of hybrid PCR devices is still being confirmed, and the problems related to sample inhibition, adsorption and bubble formation associated with the μ PCR chip approach still impose significant stringency to all previous sample preparation/nucleic acid separation methods as well as amplification reagents and reaction conditions (e.g., extremely high polymerase concentrations, PCR primer concentrations, etc.).

Summary of The Invention

One embodiment of the present invention provides a method of amplifying a template of a nucleic acid target sequence contained in a sample. The method includes contacting the sample with an amplification reaction mixture comprising a primer complementary to a template of a nucleic acid target sequence. The reaction temperature oscillates between an upper temperature limit and a lower temperature limit, wherein the temperature changes by no more than about 20 ℃ during a plurality of temperature cycles. A template of the nucleic acid target sequence is amplified.

One embodiment of the present invention provides that the temperature does not change by more than about 15 ℃ during a plurality of temperature cycles. Another embodiment provides that the temperature changes by no greater than about 10 ℃ during a plurality of temperature cycles. Yet another embodiment provides that the temperature changes by no greater than about 5 ℃ during a plurality of temperature cycles. According to one embodiment of the invention, the temperature may fluctuate (+/-2 ℃) for a given temperature and/or range.

Another embodiment of the invention provides that once the upper or lower temperature is reached, the temperature is maintained for a period of time within the temperature fluctuation. Alternatively, once an upper or lower temperature within the temperature range is reached, the temperature is changed to another temperature. In one embodiment, the lower temperature is not less than about 50 ℃. In another embodiment, the upper temperature limit is no greater than about 85 ℃. According to one embodiment, the upper and lower temperature limits may vary by about +/-5 ℃.

According to one embodiment of the invention, the template of the nucleic acid target sequence may be single-stranded DNA or RNA, double-stranded DNA or RNA, DNA or any combination thereof. The target nucleic acid can be less than 1000bp, less than 250bp, less than 150bp, or less than 100bp in length.

One or more embodiments can include a primer pair that binds to opposite strands of a nucleic acid template. The primer pair may have such a length and GC content that the melting temperature is 65 ℃ or more. In another embodiment, the primer pair has such a length and GC content that the melting temperature is 70 ℃ or higher. For example, each primer in a primer pair independently has a length of 35-70 base pairs. According to one embodiment of the invention, the melting temperature of each primer of the primer pair is 70-80 ℃. In a preferred embodiment, the primer pair comprises a forward primer and a reverse primer, each of which is 40-47 base pairs in length.

Another embodiment of the invention provides a method of amplifying a template of a nucleic acid target sequence contained in a sample. The method comprises contacting the sample with an amplification reaction mixture comprising primers or primer pairs that are 35-70 base pairs in length and are complementary to a template of the nucleic acid target sequence, and wherein each primer of the primer pair has a melting temperature of 70-80 ℃. The amplification reaction mixture also comprises DMSO, monovalent cations, divalent cations, dNTPs, and DNA polymerase. The reaction temperature oscillates between an upper temperature limit and a lower temperature limit, wherein the temperature changes by no more than about 20 ℃ during a plurality of temperature cycles, and amplifying the template of the nucleic acid target sequence. In a preferred embodiment, the divalent cation is selected from the group consisting of: magnesium, manganese, copper, zinc, or any combination thereof, and a monovalent cation selected from the group consisting of: sodium, potassium, lithium, rubidium, cesium, ammonium, or any combination thereof. In another preferred embodiment, the amplification reaction mixture comprises a nucleic acid destabilizer. In a more preferred embodiment, the reaction comprises a DNA polymerase, which may be a thermostable DNA polymerase. The DNA polymerase may be selected from the group consisting of: TAQDNA polymerase, VentR DNA polymerase, and deep ventrdna polymerase are not limited thereto as other polymerases disclosed herein and known in the art may be included. The DNA polymerase may include strand displacement activity. In another embodiment, the DNA polymerase does not have 3 '- > 5' exonuclease activity. In another embodiment, the method of amplifying a template further comprises adding a reverse transcriptase and a DNA polymerase. For example, the reverse transcriptase is a thermostable reverse transcriptase. The reverse transcriptase may be selected from: AMV-RT, Superscript II reverse transcriptase, Superscript III reverse transcriptase, or MMLV-RT, but is not limited thereto, as other reverse transcriptases known in the art may be used. Another embodiment of the invention also includes adding a single chain binding protein to the reaction mixture, as disclosed. For example, the single-chain binding protein is a thermostable single-chain binding protein or the single-chain binding protein is a non-thermostable single-chain binding protein.

Another embodiment of the invention provides a mixture comprising a single-stranded or double-stranded nucleic acid destabilizing agent. Such as dimethyl sulfoxide (DMSO) or formamide, but is not limited thereto, as other reagents such as glycerol may be added for the same purpose.

Another embodiment of the present invention provides a method wherein the sample is not alcohol-free, and or the sample is not salt-free.

Another embodiment of the invention provides a method of amplifying a template of a nucleic acid target sequence contained in a sample, wherein the amplification reaction mixture comprises a single-stranded or double-stranded nucleic acid destabilizer; a monovalent cation; a divalent cation; dNTPs and a DNA polymerase buffered at a pH that supports activity.

Another embodiment of the invention provides an amplification reaction mixture buffer comprising one or more of: single-or double-stranded nucleic acid destabilizing agents; a monovalent cation; a divalent cation; dNTPs; and a DNA polymerase buffered at a pH that supports activity. The DNA polymerase may be a thermostable DNA polymerase. The DNA polymerase may be selected from the group consisting of: TAQ DNA polymerase, VentR DNA polymerase and deep VentR DNA polymerase, but is not limited thereto. The DNA polymerase may have strand displacement activity. A DNA polymerase can be selected that does not have 3 '- > 5' exonuclease activity. The mixture may also comprise one or more of the following: single-chain binding protein, destabilizing agent is dimethyl sulfoxide (DMSO) or formamide; a divalent cation, which may be a salt selected from the group consisting of magnesium, manganese, copper, zinc, or any combination thereof; and a monovalent cation which is a salt selected from the group consisting of: sodium, potassium, lithium, rubidium, cesium, ammonium, or any combination thereof.

Objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

Brief Description of Drawings

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. The drawings are only for purposes of illustrating the preferred embodiments of the invention and are not to be construed as limiting the invention. In the drawings:

FIG. 1 shows a schematic diagram of a oscillatory PCR amplification reaction according to one embodiment of the present invention, panel A shows a trace of thermal fluctuations observed during several OPCRar cycles (grey line) compared to a conventional two-step PCR reaction (black line), and panel B shows a nucleotide amplification method according to one embodiment of the present invention.

FIG. 2, panels A-E show a series of photographs of an acrylamide gel showing different polymerases for producing a 153 base pair (bp) product according to one embodiment of the present invention.

FIG. 3, panels A-B show a series of photographs of an acrylamide gel according to an embodiment of the present invention, showing the effect of ethanol on nucleic acid amplification.

FIG. 4 is a photograph of an acrylamide gel showing the difference in annealing temperature and primer melting temperature that supports efficient amplification according to one embodiment of the present invention.

FIG. 5 is a photograph of an acrylamide gel showing the effect of hot start DNA polymerase on primer dimer formation according to one embodiment of the present invention.

FIG. 6 is a photograph of an acrylamide gel showing the effect of GC and AT clamp loops (clamp) on primer-dimer formation according to one embodiment of the present invention.

FIG. 7 is a photograph of an acrylamide gel according to one embodiment of the present invention, showing the effect of single-chain binding proteins on product formation.

FIG. 8, panels A-B show photographs of an acrylamide gel according to an embodiment of the present invention, showing that T4 gene protein 32 causes a decrease in the amount of primer-dimer formation.

FIG. 9 is a photograph of an acrylamide gel showing a specific target sequence present in amplified double-stranded DNA according to one embodiment of the present invention.

FIG. 10 is a photograph of an acrylamide gel showing the amplification of specific target sequences present in ssDNA, according to one embodiment of the present invention.

FIG. 11 is a photograph of an acrylamide gel according to one embodiment of the present invention, showing amplification of a specific target sequence present in plasmid DNA.

FIG. 12 is a photograph of an acrylamide gel according to one embodiment of the present invention, which shows amplification of single-stranded RNA.

FIG. 13 is a photograph of an acrylamide gel showing amplification of a specific target sequence in bacterial genomic DNA, according to one embodiment of the present invention.

FIG. 14 is a photograph of an acrylamide gel showing the amplification of a specific target sequence present in chloroplast NDA, according to one embodiment of the present invention.

Fig. 15 is a photograph of an acrylamide gel showing amplification of two targets sequenced according to one embodiment of the present invention.

FIG. 16 FIGS. A-B show photographs of an acrylamide gel showing amplification of a target sequence at a lower melting temperature in the presence of SSB, according to one embodiment of the present invention.

FIG. 17 FIGS. A-B show photographs of acrylamide gels showing target amplification with precise temperature control and/or rapid temperature ramping (ramping) parameters required in a typical PCR thermal cycler.

Fig. 18 is a photograph of an acrylamide gel showing amplification of target Rbcl amplified in a low cost heater without temperature swing or precise temperature control.

Detailed Description

The term "nucleic acid" as used throughout the specification and claims refers to single or double stranded DNA, RNA or DNA/RNA hybrid molecules. Single-stranded nucleic acids can have secondary structures such as hairpin, loop, and stem elements. The double-stranded or single-stranded nucleic acid may be linear or circular. The double-stranded nucleic acid may be intact or nicked. The double stranded molecule may be blunt ended or have single stranded overhang ends. The nucleic acid sample may be isolated from a cell or virus and may comprise chromosomal DNA, extrachromosomal DNA, including plasmid DNA, recombinant DNA, DNA fragments, messenger RNA, ribosomal RNA, transfer RNA, double stranded RNA or other RNA present in a cell or virus. Nucleic acids can be isolated from any number of sources, such as agriculture, food, environment, fermentation or biological fluids such as saliva, blood, nasal or lung aspirates, cerebrospinal fluid, sputum, stool, milk, swabs of mucosal tissue, tissue samples, or cells. The nucleic acid may be isolated, cloned or synthesized in vitro. Within the nucleic acids described above, individual nucleotides may be modified or chemically altered, such as methylated. These modifications or alterations may occur naturally or be produced by in vitro synthesis.

The term "target nucleic acid" or "template nucleic acid" as used throughout the specification and claims refers to a single-or double-stranded DNA or RNA fragment or sequence that is intended to be selectively amplified. The size of the nucleic acid to be amplified is defined by the upstream (5 ') and downstream (3') boundaries and may be less than 500 bp, preferably less than 250bp, and more preferably less than 150bp and more preferably less than 100 bp. The target nucleic acid may be a fragment contained within a longer double-stranded or single-stranded nucleic acid or may be an entire double-stranded or single-stranded nucleic acid.

The term "duplex" as used throughout the specification and claims refers to a DNA or RNA nucleic acid molecule that is either fully or partially double stranded.

The term "thermal cycling" as used throughout the specification and claims refers to the repetitive temperature fluctuations required for nucleic acid amplification to occur. Thermal cycling may include, but is not limited to, a high temperature melting or denaturation step, and a low temperature annealing or hybridization step.

The term "melting" or "denaturation" as used throughout the specification and claims refers to the use of high temperature to dissociate all or part of the two complementary strands of a nucleic acid duplex. The melting or denaturation temperature can be influenced by the length and sequence of the oligonucleotide primers, the concentration of duplex destabilizing agents such as DMSO and formamide, and the ionic strength or pH of the solution.

The term "annealing" or "hybridization" as used throughout the specification and claims refers to the sequence-specific binding of an oligonucleotide primer to a single-stranded nucleic acid template. The primer may bind to its complement on only one strand of the template and not to other regions of the template. The specificity of annealing or hybridization can be influenced by the length and sequence of the oligonucleotide primers, the temperature at which the binding is performed, the concentration of duplex destabilizing agents such as DMSO and formamide, and the ionic strength or pH of the solution.

As used throughout the specification and claims, the term "primer" refers to a single-stranded nucleic acid or oligonucleotide capable of binding a single-stranded region on a target nucleic acid in a sequence-specific manner, which allows for polymerase-dependent replication of the target nucleic acid.

As used throughout the specification and claims, the term "OPCRar" refers to a vibrating PCR amplification reaction, which is an in vitro technique for amplifying nucleic acids, using a temperature variation less than typical amplification techniques, e.g., less than 20 ℃, preferably less than 15 ℃, and more preferably less than 10 ℃ between the denaturation temperature and the annealing temperature.

The term "helper protein" as used throughout the specification and claims refers to any protein capable of stimulating activity, for example, a thermostable single-chain binding protein (SSB), such as rec a or RPA (replication protein a), but is not limited thereto.

In an embodiment of the invention, a method for exponential amplification of specific nucleic acid targets by thermal cycling is provided, wherein the temperature variation is preferably less than 20 ℃, more preferably less than 15 ℃, and even more preferably less than 10 ℃. This includes the following steps: providing a single-stranded template of the nucleic acid to be amplified, an oligonucleotide primer for hybridization with the nucleic acid template, synthesizing a double-stranded extension product complementary to the template strand by a DNA polymerase using the hybridized oligonucleotide primer, and repeating the above steps to exponentially amplify the selected nucleic acid target.

Referring now to FIG. 1A, a schematic diagram of a oscillatory PCR amplification reaction (OPCRar) according to one embodiment of the present invention is shown, where Panel A shows a representative trace of observed thermal fluctuations during several OPCRar cycles (grey line) compared to a conventional two-step PCR reaction (black line). Note the significant reduction in temperature variation in OPCRar. FIG. 1B shows that, according to one embodiment of the invention, a double stranded target nucleic acid enters a melting phase, wherein, depending on the temperature, partial or complete denaturation of the duplex can result. Unwinding of the duplex starts at the end of the target, and the single-stranded binding proteins (circles) bind and stabilize the single-stranded nucleic acids. The reaction is cooled and enters a hybridization/polymerization stage, in which the primers hybridize in a specific manner to the 5' end of each strand of the target duplex. After primer hybridization, a DNA polymerase (squares) binds to the template/primer duplex and replicates the template strand of DNA by extending the primer in the 5 '→ 3' direction through the introduction of dNTPs. If the polymerase used has strand displacement activity, it will be able to displace the opposite strand in the partially denatured complex. After the new duplex DNA is generated, the thermal cycle is repeated multiple times to result in exponential amplification of the target nucleic acid sequence.

In a further embodiment of the invention, the thermal cycle comprises a temperature oscillation or cycle between two temperatures, wherein Δ T is preferably no more than 20 ℃, more preferably no more than 15 ℃, and even more preferably less than 10 ℃. The higher of the two temperatures may be sufficient to denature the double stranded target DNA, or preferably only result in partial denaturation of the double stranded DNA target. Once the high or low temperature is reached, the temperature is maintained for a period of time or, preferably, immediately changed to another temperature.

In further embodiments of the invention, the nucleic acid target may be a double-stranded nucleic acid, such as double-stranded DNA, or a single-stranded nucleic acid, such as single-stranded RNA or DNA. If the target nucleic acid is double-stranded, it must be denatured, either completely or partially, by heat or with an enzyme to form a single-stranded template or template region required for DNA polymerase activity and amplification. The target nucleic acid may be less than 1000bp, preferably less than 250bp, and more preferably less than 150bp in length.

In a further embodiment of the invention, the oligonucleotide primers used for amplification of the target nucleic acid are a primer pair that binds to opposite strands of a specific double-stranded target nucleic acid, wherein one primer binds upstream of the target at the 5 'end and one primer binds downstream of the target at the 3' end. Under multiplex conditions, more than one pair of oligonucleotide primers can be used to amplify multiple nucleic acid targets simultaneously in the same reaction mixture. The oligonucleotide primers may have such a length and GC content that the melting temperature is greater than 65 ℃, preferably greater than 70 ℃ under the generally accepted PCR buffer conditions.

In a further embodiment of the invention, the DNA polymerase used is preferably selected from Taq DNA polymerase, VentR DNA polymerase, DeepVentR DNA polymerase and similar thermostable DNA polymerases. Preferably, the DNA polymerase has strand displacement activity and does not have 3 '→ 5' exonuclease activity (see fig. 1B). Furthermore, if the template nucleic acid is a single stranded RNA, the reverse transcriptase used will be selected from AMV-RT, Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.), Superscript III reverse transcriptase (Invitrogen), and similar thermostable enzymes.

Other-thermostable polymerase potential

Thermostable DNA polymerases

Polymerase and (manufacturer)

(NEB)

(NEB)

Deep Vent(NEB)

Deep VentR(exo-)(NEB)

Tag(NEB)

Pyro Script(Lucigen)

3173 and wild type (Lucigen)

LongAmp Tag(NEB)

Bst polymerase

Phire Hot Start II(NEB)

Phusion high fidelity DNA polymerase (NEB)

Phusion(NEB)

Flash(NEB)

9 Nm(NEB)

DyNAzyme II Hot Start(NEB)

DyNAzyme EXT(NEB)

DreamTag(Fermentas)

Tag (non-denatured) (Fermentas)

Hot Start Tag(Fementas)

Pfu (recombinant), (Fermentas)

Bsm (Large fragment), (Fermentas)

True StartTM Hot Start Tag(Fermentas)

Tfi(invitrogen)

(Invitrogen)

AmpliTag(Invitrogen)

Pfx

In a further embodiment of the invention, the reaction mixture preferably comprises a single-stranded binding protein (SSB), such as T4 gene 32 protein, or a thermostable SSB isolated and cloned from a thermophilic organism.

Alternatively, the enzyme preparation comprises a single-or double-stranded nucleic acid destabilizing agent such as dimethyl sulfoxide (DMSO) or formamide, preferably in a concentration of 8-15% of the total reaction volume. Alternatively, other agents such as glycerol deaza-dGTP, 3 dazopurine, dITP may be used alone or in combination with each other or the agents listed above.

Embodiments of the present invention are ideally suited for low cost, point-of-care nucleic acid diagnostics where a microfluidic flow layer is placed over a heating element. By reducing the temperature range cycling requirements, a relatively simple heating mechanism with passive cooling can be used to rapidly cycle the temperature of the reaction solution. The lower maximum temperature during thermal cycling minimizes fluid evaporation (which can negatively impact the overall amplification reaction). More importantly, the robustness of the amplification is greatly improved compared to conventional PCR methods, suggesting that the new method can accommodate temperature fluctuations during the amplification process (imprecise temperature control). It is demonstrated that the specific reaction chemistry of the present invention proceeds over a wide melting (e.g., 70-105 ℃, substantially insensitive to foaming) and hybridization temperature range, thereby eliminating the need for uniform temperatures throughout the reaction volume. Finally, embodiments of the invention perform well in the presence of alcohols and salts (e.g., -10% ethanol), greatly reducing the stringency of previous nucleic acid isolation methods by eliminating the centrifugation, hot drying, or vacuum steps between the alcohol-based wash (e.g., ethanol or isopropanol) and nucleic acid elution steps involved in conventional nucleic acid extraction chemistry.

Embodiments of the invention include detecting a pathogen in a biological sample, wherein the nucleic acid of the pathogen is a target nucleic acid. Alternatively, the present invention can be used to detect a difference in chromosomal DNA in which a fragment of the chromosomal DNA is a target nucleic acid. In this way, single nucleotide polymorphisms can be detected in target nucleic acids from the same or different sources.

An embodiment of the amplification technique of the present invention is referred to as the "oscillation PCR amplification reaction" (OPCRar). OPCRar is based on, but is not limited to, the use of a double-stranded destabilizer that lowers the melting temperature of the reaction in combination with an oligonucleotide primer of very high melting temperature (Tm) to raise the annealing temperature in a given thermal cycle. In this way, in vitro amplification of a target nucleic acid can be performed by rapid thermal cycling between temperatures that preferably differ by 20 ℃, more preferably differ by less than 15 ℃, and even more preferably differ by less than 10 ℃ (fig. 1A). Oligonucleotide primers specific for the upstream (5 ') and downstream (3') regions of the target nucleic acid hybridize to the template, allowing for amplification of the target by extension with a DNA polymerase. If the DNA polymerase used is a strand displacing polymerase without exonuclease activity, complete thermal denaturation of the double stranded target nucleic acid is not necessary, acting in conjunction with a duplex destabilizing agent to lower the desired melting temperature. The temperature cycling process is repeated and results in exponential amplification of specific nucleic acid target sequences (fig. 1B). In OPCRar, a double stranded target nucleic acid enters a melting phase, where, depending on the temperature, partial or complete denaturation of the duplex can result. Unwinding of the duplex starts at the end of the target, and the single-stranded binding proteins (circles) bind and stabilize the single-stranded nucleic acids. The reaction is cooled and enters a hybridization/polymerization stage, in which the primers hybridize in a specific manner to the 5' end of each strand of the target duplex. After primer hybridization, a DNA polymerase (squares) binds to the template/primer duplex and replicates the template strand of DNA by extending the primer in the 5 '→ 3' direction through the introduction of dNTPs. If the polymerase used has strand displacement activity, it will be able to displace the opposite strand in the partially denatured complex. Once new duplex DNA is generated, thermal cycling is repeated multiple times to result in exponential amplification of the target nucleic acid sequence.

The OPCRar method is based on, but not limited to, the combined use of a nucleic acid destabilizer that lowers the melting temperature of the reaction and two oligonucleotide primers of very high melting temperature (Tm) to raise the annealing temperature during thermal cycling. For a given target nucleic acid, one oligonucleotide primer preferably hybridizes to the 5 '-end of the sense strand containing the target sequence and one primer preferably hybridizes to the 5' -end of the antisense strand containing the reverse complement of the target sequence. OPCRar preferably utilizes, but is not limited to, the use of a strand-displacing DNA polymerase without exonuclease activity to further reduce the melting or denaturation temperature required for efficient target nucleic acid amplification. OPCRar can amplify a target nucleic acid in the presence or absence of a helper protein. Any specific OPCRar system can be optimized by adding, subtracting or replacing components within the mixture.

In the context of low cost, rapid, point-of-care nucleic acid diagnostics, this amplification technique has improved properties over other amplification methods reported in the prior art. Unlike the nucleic acid amplification methods described above, the OPCRar system, the robust enzymatic process of which enables robust, fast, and temperature fluctuation tolerant heating of low-cost heating equipment, is therefore ideally suited for low-cost, point-of-care applications. OPCRar combines the speed and reliability of PCR with the reduced equipment requirements of isothermal amplification methods by minimizing the temperature differences encountered during thermal cycling.

The simplified thermal cycling requirements of the OPCRar system are ideally suited for passive-cooling arrangements, where heat can be applied to one surface of the chamber and cooling occurs on the opposite surface by dissipating the heat into the air. Such passive-cooling greatly reduces the cost and complexity of any nucleic acid diagnostic device. Passive-cooling has been previously reported for diagnostic devices, however, these devices employ conventional PCR cycling assay chemistry to amplify the target nucleic acid, which limits the rate of the reaction (Luo et al NucAcids Res.2005; Wilding et al, Nuc.acids.Res.1996; Burke et al, Science 1998;). Another advantage of OPCRar is that efficient nucleic acid amplification can occur over a wide melting and annealing temperature range and therefore requires less stringent temperature control mechanisms. In the construction of miniaturized nucleic acid diagnostics, it can be challenging to maintain a uniform temperature throughout the reaction volume, with a high temperature gradient existing between the heated and unheated sides of the reaction chamber. Such temperature changes may result in inefficient amplification using conventional PCR or isothermal reaction chemistry. OPCRar, designed to minimize problems associated with precise temperature regulation and maintenance by using a combination of robust polymerases, destabilizers, and other polymerase cofactors; as long as the coolest region of the reaction chamber complies with the lowest possible melting temperature and the highest possible annealing temperature for a given nucleic acid target reaction, the reaction will proceed efficiently, even if other regions of the reaction volume vary >10 ℃. furthermore, the robust OPCRar nature of the amplification chemistry with minimal power/energy consumption enables fast and efficient amplification reactions in larger volumes (e.g. 20 μ Ι instead of below μ Ι in typical μ tpa chips), this greatly relaxes the stringency of the previous sample preparation/nucleic acid separation process (in terms of obtaining input templates of μ l or less, which are highly concentrated and ultrapure nucleic acids free of any trace contaminants such as salts and ethanol residues and inhibitory substances) and the requirement of ultra-high concentrations and ultra-purity of PCR enzymes and biological reagents.

Solvent reagent

Solvents such as DMSO and formamide are known to lower the melting temperature of duplex nucleic acids by-0.5-0.7 deg.C (per 1% volume added). These reagents are typically used to increase the amplification efficiency of target nucleic acids containing high GC content and, therefore, high Tm to promote complete denaturation of double stranded templates that are difficult to unwind. Typically, the PCR thermal cycling temperature is maintained after the duplex destabilizer is introduced into the PCR reaction. In contrast, OPCRar preferably utilizes the addition of DMSO at uniquely high concentrations to significantly reduce the melting temperature of thermal cycling. In conventional PCR, DMSO is rarely used at greater than 10% v/v due to the loss of polymerase activity associated with high concentrations of these reagents and high temperatures at the melting stage (typically greater than 90 ℃). OPCRar systems and methods, on the other hand, preferably use DMSO concentrations of 10 to 15%. Surprisingly, this amount of DMSO did not result in significant loss of polymerase activity.

Referring now to FIG. 2, amplification according to one embodiment of the invention is compatible with a variety of DNA polymerases, depending on the conditions. Ribonucleic acid (0.3 ng/. mu.tL) isolated from nasal aspirates containing influenza A virus was used as a nucleic acid template under multiple reverse transcriptase-OPCRar conditions using VentR polymerase. OPCRar primers FP3 and RP4 were used to generate 153bp products that were visualized by electrophoresis on a 12% polyacrylamide gel and staining with ethidium bromide. All reactions contained Superscript III reverse transcriptase, with an initial cDNA generation phase performed at 55 ℃ for 5 minutes, followed by thermal cycling. The gel lanes are labeled with DMSO concentration, and the melting and hybridization stage temperatures used; the reaction was cycled 40 times.

Conventional PCR enzymes, such as Taq DNA polymerase reaction mixtures, are extremely sensitive to any trace amount of alcohol, such as ethanol, whereas in one embodiment of the invention, the novel reaction mixtures are extremely resistant to inhibition by ethanol. Referring now to FIG. 3, the effect of ethanol on nucleic acid amplification is shown, according to one embodiment of the present invention. Total nucleic acid (3.4 ng/. mu.L) isolated from Asian species (Candidatus Liberibacter asiaticus) -infected leaf tissue of the species Pholiopsis citricola was used as a priming template. The reaction was performed under OPCRar conditions using VentR (exo-) DNA polymerase, Et SSB, and primers hygl _ For and hygl _ Rev, in the presence of 15% DMSO (FIG. 3A), or under conventional PCR conditions using Taq polymerase and primers HLBas-P2 and HLBr-P1 (FIG. 3B). The OPCRar solution was heated at 85 ℃ for 2 min to denature the template and then cycled 40 times with shaking between 76 ℃ for 10 sec and 60 ℃ for 10 sec to yield a 139 bp product. The conventional PCR reaction was heated to 95 ℃ for 2 minutes and then cycled 40 times, varying between 95 ℃, 10 seconds and 58 ℃, 40 seconds to generate a 130 bp product. The amplified products were visualized on a 12% acrylamide gel and stained with ethidium bromide. Showing the concentration of ethanol contained in the amplification reaction mixture. It is evident that OPCRar preparations (fig. 3A) are significantly more resistant to inactivation by ethanol than conventional PCR (fig. 3B).

Using VentR (exo-) DNA polymerase and Et SSB under typical OPCRar conditions, no loss of activity occurred up to 10% ethanol. This is a significant but surprising finding regarding the application of OPCRar to low cost, point of care devices. Since conventional knowledge of PCR and isothermal amplification typically suggests that the user provide a highly purified alcohol-and salt-free input of nucleic acid. As a result, almost all researchers have employed some type of vacuum drying, air drying, spin down (spin down) or heating step between alcohol-based washing and elution of target nucleic acids from nucleic acid affinity beads, glass frit, matrices, or filters, etc., prior to their PCR amplification process on a sample. For the example of an integrated point-of-care diagnostic device, in addition to amplifying and detecting nucleic acids, the device must also rapidly isolate target nucleic acids. Typically, this is done by: nucleic acids are bound to a glass fiber matrix and washed in the presence of significant concentrations of salt and ethanol, followed by elution in a buffer containing minimal salt and no ethanol. Prior to elution, the wash buffer remaining on the binding matrix is removed to prevent conversion to an elution volume; in commercial nucleic acid isolation kits, this is typically done by centrifugation. The specific enzyme mixture of OPCRar does not require careful removal of ethanol during nucleic acid isolation, making this embodiment of the invention suitable for low cost integrated diagnostics that do not require vacuum, centrifuge, air drying or heat drying components.

Primer and method for producing the same

The oligonucleotide primers described herein can be synthesized and purified by methods known in the art. (see, e.g., U.S. patent No. 6,214,587). In this embodiment, two sequence-specific primers representing a primer pair are used to exponentially amplify a target nucleic acid sequence. The first primer hybridizes to the upstream 5 'region of the target nucleic acid and the second primer hybridizes to the downstream, 3' region of the target sequence. The primer hybridizes to the 5 ' end of one strand present in the target duplex and the primer is extended in the 5 ' to 3 ' direction by a polymerase using the target nucleotide sequence as a template (fig. 1B). The conditions for hybridization are as described in "Molecular cloning and Laboratory Manual", 2nd ed.Sambrook, Rich and Maniatis, pub.Cold Spring Harbor (2003). To achieve specific amplification of a given target sequence, homologous primers are preferred, wherein each nucleotide in the primer is complementary to the target sequence. However, the primer may include a base that is not homologous to the template nucleic acid, or a 5' sequence that is not complementary to the target nucleotide sequence. Multiple pairs of primers can be used in a single OPCRar experiment to simultaneously amplify multiple nucleic acid targets in the same reaction mixture. The so-called multiplex method (multiplexing) is a technique generally used in single nucleotide polymorphism analysis, detection of pathogens, and binding of internal controls to individual reactions. High level multiplexing can also be achieved by using 5 'universal tag sequences introduced into individual target-specific 3' regions that allow universal amplification of all target sequences with different internal pathogen sequences.

Oligonucleotide primer design involves several parameters such as melting temperature and intra-and inter-primer sequence alignments. The melting temperature is controlled by factors such as primer length and GC content. Complementarity between primer sequences may result in hairpin structures that may prevent efficient amplification, while homology within the primers may result in undesired amplification products replicating primer-dimers. In designing primers, it is important to select within the target a sequence that is specific for the nucleic acid molecule to be amplified and that will minimally interact with itself or other primers present in the amplification reaction.

In most nucleic acid amplification strategies, the melting temperature of the primers is preferably about 10 to 30 ℃ higher than the temperature at which hybridization and amplification occur. In the case where the temperature of the annealing/polymerisation stage in the PCR reaction is 55-60 ℃, the length of the primer is typically 18-30 base pairs. The specific oligonucleotide is made to be the shortest length to allow easy primer binding without loss of sequence specificity. However, in the OPCRar system, the primers are preferably designed to be very long 35-55 base pairs, and the melting temperature is preferably 70-80 ℃ in order to increase the temperature during the annealing phase. Given the level of duplex destabilizer DMSO used in a typical OPCRar reaction (-10-15%), the calculated Tm for the OPCRar primers is preferably only <10 ℃ above the annealing temperature used during thermal cycling. In experiments and with extreme lengths of OPCRar primers, efficient amplification occurred despite the minimal difference in primer Tm (offset DMSO concentration) and annealing/extension temperatures.

Referring now to FIG. 4, in accordance with one embodiment of the invention, OPCRar primers require minimal differences between annealing stage temperature and primer Tm to support efficient amplification. The plasmid containing the hyvl gene sequence (12 ng/. mu.L) was amplified using primers hyvl _ For and hyvl _ Rev to generate 139 bp products, which were visualized by electrophoresis on a 12% acrylamide gel, staining with ethidium bromide. After the initial 2 min 85 ℃ melting step, all reactions were cycled 40 times, 10 seconds each at the indicated melting and hybridization temperatures. The Tm For primers hyvl _ For and hyvl _ Rev were calculated to be 72.2 ℃ and 70.9 ℃ respectively. The reaction was performed in 10% DMSO, decreasing the effective Tm by 7 ℃, assuming a 0.7 ℃ decrease per 1% volume. Even with negligible difference between primer Tm and hybridization temperature, the amplification reaction was observed to proceed as efficiently as the temperature difference was much lower.

Referring now to FIG. 5, the effect of hot start DNA polymerase on primer dimer formation using OPCRar. Control reactions without nucleic acid template were performed using Superscript III RT and Taq or Platinum Taq DNA polymerase in the presence of the primer pair FP3 and RP4 (8. mu.M each) and 15% DMSO. The cycle parameters were 55 ℃ for 5 minutes, 85 ℃ for 2 minutes, and 80 ℃ for 15 seconds and 65 ℃ for 15 seconds for 40 cycles. After electrophoresis on a 12% acrylamide gel, the OPCRar product was visualized by staining with ethidium bromide. Platinum Taq is a commercially available hot start enzyme (Invitrogen, Carlsbad, CA) conjugated to antibodies that dissociate under normal PCR conditions after heating the reaction solution to 94 ℃. In the presence of the template nucleic acid, this primer pair will yield a 153bp product. Clearly, the hot start formulation is the same as regular Taq in reducing <110 bp primer dimer formation during OPCRar. One complication of the long primers used in the OPCRar reaction is that they are more prone to produce non-specific and undesired amplification products called primer dimers. Primer dimers are formed when the 3' end of the primer oligonucleotide transiently binds to one another during an initial temperature increase at the beginning of the amplification reaction. During this critical time, the DNA polymerase can extend these transient complexes to produce products that compete with the specific target amplification during thermal cycling, especially if the initial template nucleic acid concentration is very low. A technique commonly used to reduce primer dimer formation during PCR is the use of so-called "hot start" DNA polymerases. These commercially available enzymes are non-covalently bound to inhibitory molecules such as antibodies. When the reaction temperature is increased above 90 ℃, the molecules are inhibited from dissociating, releasing the polymerase to function normally. However, surprisingly, in our hands, the hot start enzyme Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) failed to considerably reduce the abundance of primer dimer amplification, indicating that this commonly used method is not sufficient for OPCRar, indicating that the OPCRar method is a much more efficient amplification method than conventional PCR, which enables dimer formation resulting from homo-and heterodimer transient kinetic collisions, which typically do not occur in conventional PCR methods.

Referring now to FIG. 6, presented is a gel showing the effect of GC and AT clamp loops on primer dimer formation during OPCRar according to one embodiment of the present invention. Primers designed to amplify the elongation factor gene of the Asian species of the Pholiopsis citricola were used in the absence of a priming template (3.5 ng/. mu.L) to determine the propensity for primer dimer formation. In the presence of the template, the product size of the different primer sets will be 140-155 bp. Primer sequences can be seen on the right, with AT or GC clamp loops in grey. For the "mod" primer set, each primer contains a different base from the template (underlined), which increases homology to the second primer. All reactions were performed using VentR (exo-) DNA polymerase, Et SSB, in the presence of 15% DMSO. The solution was heated at 85 ℃ for 2 minutes to denature the template and then cycled 30 or 40 times, shaking between 80 ℃ for 15 seconds and 65 ℃ for 15 seconds. The amplified products were visualized on a 12% acrylamide gel stained with ethidium bromide. As can be clearly seen, the GC clamp primer set resulted in significant primer dimer formation, whereas the AT clamp primer did not result in primer dimer formation.

To minimize the possibility of primer dimer formation, OPCRar primers can be designed to employ several strategies distinct from those used to generate conventional PCR primers. First, PCR primers typically have a GC-rich 3' end called a "GC clamp loop" which results in greater specificity of binding to the target sequence. However, in the OPCRar primer, it was observed that high GC content in the 3 'region of the primer resulted in more primer dimer formation, therefore, the OPCRar primer was made to contain an AT-rich 3' region to reduce the affinity of the 3 '-3' primer interaction in terms of energy to produce these undesired amplification products (fig. 6). A second strategy for OPCRar primer design is to design a primer comprising a complementary 5' or internal sequence of at least 5 contiguous nucleotides. Oligonucleotides designed in this way direct any primer hybridization during the initial increase in reaction temperature to duplex structures that are not competitive for polymerase extension. Non-homologous or mutated bases can be used to generate OPCRar primers if no suitable complementary sequence can be found within the target nucleic acid sequence. The abnormal length of the OPCRar primers overcomes the slight mismatch between the primer and the target during the early cycles of the amplification reaction. The primer group is

EU AT

Forward direction

GTTCTTGTAG CGTTGCAGTC TTCTGCGGAA GATAAGGAAT TGCTTT (SEQ ID NO 21) reverse

GGGCACGTTT ATTAGCAACA ATAGAAGGAT CAAGCATCTG CACAGAAAT(SEQ ID NO 22)EU GC

Forward direction

CTTGTAGCGT TGCAGTCTTC TGCGGAAGAT AAGGAATTGC TTTCTGCG (SEQ ID NO 23) reverse direction

CACGTTTATT AGCAACAATA GAAGGATCAA GCATCTGCAC AGAAATCACCG(SEQ ID NO 24)

EU—Atmod

Forward direction

GGTGTTCTTG TATCGTTGCA GTCTTCTGCG GAAGATAAGG AATTGCTTT(SEQ ID NO 25)

Reverse direction

GTAATGGGCA CGTTTATTAG CAACGATAGA AGGATCAAGC AACTGCACAG AAAT(SEQ ID NO26)

EU GCmod

Forward direction

CTTGTATCGT TGCAGTCTTC TGCGGAAGAT AAGGAATTGC TTTCTGCG(SEQ ID NO 27)

Reverse direction

GGCACGTTTA TTAGCAACGA TAGAAGGATC AAGCATCTGC ACAGAAATCA CCG(SEQ ID NO28)

The OPCRar primers can include any of the deoxyribonucleotide bases adenine "A", thymine "T", guanine "G" or cytosine "C" and/or one or more ribonucleotide bases A, C, uracil "U", G. In addition, the OPCRar primer may comprise one or more modified deoxyribonucleotide or ribonucleotide bases wherein the modification does not prevent hybridization of the primer to a target nucleic acid, primer extension by a polymerase, or denaturation of duplex nucleic acids. The OPCRar primer may be modified with a chemical group such as methyl phosphonate or phosphorothioate, a non-nucleotide linker, biotin, or a fluorescent label such as an amine-reactive fluorescein ester of carboxyfluorescein. These modifications may enhance primer performance or facilitate detection and characterization of amplification products.

Polymerase enzyme

The polymerization step occurs after the single-stranded template nucleic acid region is hybridized to the primer during OPCRar. If the target nucleic acid is DNA, a DNA polymerase is selected which acts on the target to extend the hybridized primer along the nucleic acid template in the presence of the four dNTP nucleotide bases to form a double stranded product in which the newly synthesized strand is complementary to the nucleotide sequence of the template (FIG. 1). If the initial target is RNA, reverse transcriptase is first used to copy the RNA template into the cDNA molecule, which is further amplified by DNA polymerase during OPCRar.

A variety of DNA polymerases can be selected for OPCRar based on thermostability and processivity (especially in the presence of destabilizing agents and alcohols) (FIG. 2). Although not required, it was found that a polymerase exhibiting strand displacement activity and lacking exonuclease activity significantly improved the OPCRar reaction (fig. 2). Examples of suitable DNA polymerases include Taq polymerase, KlenaTaq DNA polymerase (AB Peptides, (St Louis, MO)), Bst DNA polymerase large fragment (New England Biolabs, Beverly, MA), VentR or VentR (exo-) (New England Biolabs), DeepVentR or DeepVentR (exo-) (New England Biolabs), and the like. Suitable thermostable reverse transcriptases include Superscript II (Invitrogen, Carlsbad, Calif.), Superscript III (Invitrogen), and similar enzymes. It should be noted that the published conventional PCR amplification polymerase mixtures were not capable of performing OPCRar due to the unique robustness requirements of OPCRar amplification. All selected polymerase and biological reagent components should be carefully evaluated and experimentally tested prior to use.

Single chain binding proteins

The OPCRar system is preferably such that the temperature difference during the thermal cycling phase of melting and annealing is minimized, wherein the temperature difference is minimal if complete denaturation of the duplex nucleic acid is not necessary. Strand displacement DNA polymerases are useful in this regard, while helper proteins can be used to further reduce the heat requirement for efficient amplification. Single-stranded binding proteins (SSBs) are known to stabilize single-stranded nucleic acids to prevent double-stranded annealed duplex formation, and have been shown to increase the efficiency of nucleic acid amplification reactions. It was found that the addition of thermostable SSB to OPCRar process according to embodiments of the present invention resulted in increased activity (fig. 7). As an example, Et SSB (BioHelix Corporation, Beverly, MA), although the choice of SSB is not limited to a particular protein and may include SSB isolated and cloned from a thermophilic organism, or SSB engineered from a non-thermostable precursor SSB.

Referring now to FIG. 7, illustrated is a gel showing the effect of single chain binding proteins on the formation of OPCRar products, according to one embodiment of the present invention. Universal influenza a single stranded DNA template (1E6 copies/. mu.l, Biosearch Technologies, inc., Novato, CA) was amplified using OPCRar primers FP3 and RP3 to yield a 133 bp product that was visualized by electrophoresis on a 12% acrylamide gel, staining with ethidium bromide. In the presence or absence of thermally stable SSB, under the following conditions: i) 15% DMSO; ii) 15% DMSO, 5% glycerol; iii) 15% DMSO, 0.25M Betaine, for OPCRar according to one embodiment of the invention. The cycling parameters for all reactions were 75 ℃ for 15 seconds and 65 ℃ for 15 seconds, repeated 45 times.

In addition to thermostable SSBs that aid OPCRar, non-thermostable SSBs such as T4 phage SSB (newengland biolabs) can be used to reduce primer dimer formation during initial heating of OPCRar solutions (fig. 8). By pre-incubating OPCRar primers in the presence of a molar excess of T4 gene 32 protein and then adding them to the reaction mixture, it has been observed that amplification of undesired primer dimers is minimized during OPCRar. These SSBs may bind to single-stranded oligonucleotide primers, thereby reducing the likelihood of 3 '-3' pairing and, therefore, reducing primer dimer formation. Upon heating the solution above 65 ℃, T4 SSB was denatured and released the primers for normal reactivity during thermal cycling. Referring now to FIG. 8, the gel shows a reduction in the amount of primer-dimer formed during one embodiment of the invention by pre-incubating the primer with T4 gene 32 protein. The OPCRar primers were incubated with the indicated stoichiometric excess of activity units of T4 gene 32 protein (T4 SSB) at 25 ℃ in the presence of 1X thermolpol buffer (New England BioLabs, Beverly, MA) for 5 minutes prior to addition to the reaction mixture. Synthetic universal influenza a DNA template (1E6 copies/. mu.l, Biosearch Technologies, Inc.) was amplified using primers FP3 and RP3 to yield a product sequence of 133 bp. The reaction was held at 85 ℃ for 2 minutes, followed by 50 cycles of 75 ℃ for 15 seconds and 65 ℃ for 15 seconds. The reaction products were visualized by electrophoresis on a 12% acrylamide gel, stained with ethidium bromide. The experiment described in figure 7 was repeated by different investigators on different days to assess the reproducibility of the data and is shown in figure 8. It is clearly visible that preincubation of the primers with T4 SSB increased the amount of amplified product and decreased the intensity of the primer-dimer band (. about.100 bp) compared to no preincubation (right-most lane).

The OPCRar method is particularly well suited for use with devices such as those described in a commonly owned provisional patent application entitled "integrated device for nucleic acid detection and identification" filed on even date herewith. The structure of some embodiments of the apparatus enables rapid cycling of the solution temperature while the solution remains in the same chamber, preferably without active cooling. For example, the temperature may be sufficiently increased or decreased to conduct OPCRar in less than or equal to 20 seconds, or, more preferably, less than or equal to 15 seconds, or, even more preferably, less than or equal to about 8 seconds, or, even more preferably, less than or equal to about 4 seconds. Thus, OPCRar temperature cycling can be performed in as little as 8 seconds or even faster.

Example 1: method for amplifying DNA target duplexes by OPCRar

To confirm that OPCRar is capable of amplifying a specific target sequence present in a double-stranded DNA analyte, we generated a 140 bp sequence from a PCR amplified fragment of the Asian elongation factor gene of Xanthomonas citri by the OPCRar system using two OPCRar primers, primer HLB (Huang Long bing) ForSh and primer HLBRevSh. OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by mixing:

8.4 μ L of water

2.0 μ L10 XOPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer set HLBForSh and HLBRevSh (4 μ M each)

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

2.0 μ L of PCR product dilutions (0.6 to 0.0006 ng/. mu.L starting concentration)

The reaction was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 5 seconds at 80 ℃ and 5 seconds at 65 ℃. After completion of the reaction, 5 μ L of OPCRar product was mixed with 2 μ L of 6X loading buffer (New England BioLabs) and 1 μ L of formamide, electrophoresed on a 12% acrylamide gel, and visualized with ethidium bromide. The 140 bp product was clearly observed in all dilutions shown and matched the expected length of the OPCRar target sequence (figure 9).

Referring now to FIG. 9, amplification of a specific target sequence present in double-stranded DNA according to one embodiment of the invention is shown in a gel. A serial dilution (0.6 to 0.0006 ng/. mu.L) of PCR amplified fragments of the Asian elongation factor gene of the species of Xanthomonas citri was used as the initial template. Reactions were carried out in the presence of 15% DMSO using VentR (exo-) DNA polymerase, Et SSB, and primers HLBForSh and HLBRevSh to generate a 140 bp sequence. The reaction was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 5 seconds at 80 ℃ and 5 seconds at 65 ℃. OPCRar products were visualized on 12% acrylamide gels stained with ethidium bromide. A140 bp product matching the expected length of the target sequence was clearly observed in all dilutions shown.

Example 2: method for amplifying single-stranded DNA target by OPCRar

To confirm that OPCRar is able to amplify specific target sequences from single stranded DNA templates, we used OPCRar primers FP3 and RP4 by the OPCRar system to generate 153bp sequences from a commercially available universal influenza a template (Biosearch Technologies, Inc.). The 10XOPCRar buffer contained 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100. Prepare 20 μ L of OPCRar solution by mixing:

8.4 μ L of water

2.0 μ L of 10x OPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer sets FP3 and RP4 (8 μ M each)

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

mu.L of single-stranded DNA template (1E9 copies of 1E 2/. mu.L).

For sensitivity comparison, real-time PCR reactions were performed using the same template concentrations as used for OPCRar above. 10 XThermoPol (New England BioLabs) contained 200 mM Tris-HCl (pH 8.8), 100 mM ammonium sulfate, 100 mM potassium chloride, 20 mM magnesium sulfate, and 1% Triton X-100. Prepare 15 μ L RT-PCR solution by mixing:

9.7 μ L of water

1.5 μ L10x ThermoPol buffer

0.4μLdNTPs(10 mM)

1.5 μ L of primer set UniAfCDC/UniArCDC (4 μ M each), including TaqMan probe UniApCDC (1 μ M)

0.4 μ L Taq polymerase (5U/. mu.L)

1.5 μ L Single-stranded DNA template (1E 9-1E 2 copies/. mu.L)

Serial dilutions of 1E9 to 1E2 copies/. mu.l of universal influenza a single stranded DNA template were amplified by OPCRar in the presence of 15% DMSO and real-time PCR was performed using TaqMan probes. The OPCRar reaction was first heated to 85 ℃ for 2 minutes and then cycled between 80 ℃ for 15 seconds and 65 ℃ for 15 seconds, repeating 40 times. The RT-PCR reaction was heated to 95 ℃ for 2 minutes and then cycled 45 times between 10 seconds at 95 ℃ and 40 seconds at 58 ℃. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2. mu.L of 6X loading buffer (New England BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. For all samples, a 153bp product was clearly observed (fig. 10) and matched the expected length of the OPCRar target sequence.

Referring now to FIG. 10, presented is a gel showing target sequences present in a single stranded DNA template amplified according to one embodiment of the present invention. Serial dilutions of universal influenza a single stranded DNA template (1E9 to 1E2 copies/. mu.l, Biosearch Technologies, Inc.) were amplified by OPCRar in the presence of 15% DMSO using primers FP3 and RP4 to produce 153bp products that were visualized by electrophoresis on a 12% acrylamide gel using ethidium bromide staining (left panel). For comparison, the same dilution was used as the initial template for real-time PCR, using primer sets uniaffcdc/uniartcdc and TaqMan probe UniApCDC (right panel). The OPCRar reaction was first heated to 85 ℃ for 2 minutes, then cycled between 80 ℃ for 15 seconds and 65 ℃ for 15 seconds, repeating 40 times. The RT-PCR reaction was heated to 95 ℃ for 2 minutes and then cycled 45 times between 10 seconds at 95 ℃ and 40 seconds at 58 ℃. It is evident that OPCRar, when suitably optimized, has a sensitivity similar to that of a conventional PCR reaction.

Example 3: method for amplifying specific sequences present on plasmid DNA by OPCRar

To confirm that OPCRar is able to amplify specific target sequences present in double-stranded plasmid DNA, we generated 139 bp sequences from a plasmid containing the hyvl gene of liberibacter citreus asian species by the OPCRar system using two OPCRar primers, primer hyvl _ For and primer hyvl _ Rev. OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by mixing:

9.4 μ L of water

2.0 μ L of 10x OPCRar buffer

2.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer sets hyvl _ For and hyvl _ Rev (8 μ M each)

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

2.0 μ L of DNA extracted from healthy and Citrus flavedo-infected tissue (17.2 ng/. mu.L)

Titration was performed with DMSO (13-8% v/v). The reaction was heated at 85 ℃ for 2 min to denature the template and then cycled 40 times, shaking between 80 ℃ for 10 sec and 65 ℃ for 10 sec. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2L of 6X loading buffer (New England BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. For all samples, a 139 bp product was clearly observed (fig. 11) and matched the expected length of OPCRar target sequence.

Referring now to FIG. 11, a gel of specific target sequences present in plasmid DNA amplified according to one embodiment of the present invention is presented. A plasmid containing the Asian species hygl gene of the species Phomopsis citricola (17.2 ng/. mu.L) was used as the initial template and the concentration of DMSO (13-8% v/v) was titrated. All reactions were performed using VentR (exo-) DNA polymerase, Et SSB, and primers hygl _ For and hygl _ Rev. The reaction was heated at 85 ℃ for 2 min to denature the template and then cycled 40 times, shaking between 80 ℃ for 10 sec and 65 ℃ for 10 sec. OPCRar products were visualized on 12% acrylamide gels stained with ethidium bromide. At all DMSO concentrations tested, a 139 bp product matching the expected length of the target sequence was clearly observed.

Example 4: method for amplifying RNA target sequence of human pathogenic virus present in nasal aspirate by OPCRar

To confirm that OPCRar is able to amplify specific target sequences present in single stranded RNA templates, we generated 153bp sequences from ribonucleic acids isolated from clinical nasal aspirates infected or not infected with influenza a virus using OPCRar primer pair, FP3 and RP 4. OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by combining:

9.3 μ L of water

2.0 μ L10 XOPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer sets FP3 and RP4 (8 μ M each)

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

0.1 μ LSuperscript III reverse transcriptase (200U/. mu.L)

2.0 μ L nucleic acid isolated from clinical nasal aspirate (0.3 ng/. mu.L)

The reaction was incubated at 55 ℃ for 5 minutes to generate cDNA, heated to 85 ℃ for 2 minutes to denature the template and then cycled 40 times, shaking between 80 ℃ for 10 seconds and 65 ℃ for 10 seconds. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2. mu.L of 6X loading buffer (New England BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. The 153bp product was clearly observed in positive but not negative clinical samples (fig. 12) and matched the expected length of OPCRar target sequence.

Referring now to FIG. 12, illustrated is a gel showing specific target sequences present in single stranded RNA amplified according to one embodiment of the present invention. Ribonucleic acid (0.3 ng/. mu.L) isolated from nasal aspirates infected or not infected with influenza A virus was used as template. All reactions were performed in the presence of 15% DMSO using Superscript III reverse transcriptase, VentR (exo-) DNA polymerase, Et SSB, and primers FP3 and RP 4. The reaction was incubated at 55 ℃ for 5 minutes to generate cDNA, heated to 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 80 ℃ for 10 seconds and 65 ℃ for 10 seconds. OPCRar products were visualized on 12% acrylamide gels stained with ethidium bromide. A153 bp product of the expected length matching the target sequence was clearly observed in positive but not negative clinical samples.

Example 5: method for amplifying target sequence from pathogenic plant bacterium by OPCRar

To confirm that OPCRar is capable of amplifying specific target sequences present in the genome of pathogenic bacteria, we generated a 213 bp fragment of the Asian elongation factor gene of Phosphaera citricola from total nucleic acid isolated from infected plant tissue using the OPCRar primer pair, EU523377-F-57 and EU 523377-R-56. An OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by combining:

8.4 μ L of water

2.0 μ L of 10x OPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer set EU523377-F-57 and EU523377-R-56 (4 μ M each), primer EU523377-F-57 being biotinylated (5') or non-biotinylated

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

2.0 μ L Total nucleic acid isolated from Asian infected plant tissue of the species Pholiopsis citriodora (1.1 ng/. mu.L)

To demonstrate that primer modification is suitable for OPCRar, the forward primer EU523377-F-57 was biotinylated at the 5' end in some reactions. The OPCRar solution was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times, shaking between 80 ℃ for 15 seconds and 65 ℃ for 15 seconds. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2. mu.L of 6X loading buffer (NewEngland BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. The 213 bp product was clearly observed in all samples and matched the expected length of the OPCRar target sequence (fig. 13).

Referring now to FIG. 13, presented is a specific target sequence present in bacterial genomic DNA as amplified according to one embodiment of the invention. Total nucleic acid isolated from Asian infected leaf tissue (1.1 ng/. mu.L) of the species Xanthomonas citri was used as the initial template. All reactions were carried out in the presence of 15% DMSO using VentR (exo-) DNA polymerase, Et SSB, and primers EU523377-F-57 and EU 523377-R-56. Primer EU523377-F-57 is biotinylated or not biotinylated at the 5' end of the oligonucleotide. The OPCRar solution was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times, shaking between 80 ℃ for 15 seconds and 65 ℃ for 15 seconds. The amplified products were visualized on a 12% acrylamide gel stained with ethidium bromide. For all positive samples but not negative samples, a 213 bp product was clearly observed, which matched the expected length of the OPCRar target sequence.

Example 6: method for amplifying specific sequence on organelle DNA

To confirm that OPCRar is able to amplify specific target sequences present in organelle DNA (in this case chloroplast DNA), we generated a 137 bp fragment of the rbcL gene of plants from total nucleic acid isolated from plant tissues infected or not infected with liberibacter asiaticus using OPCRar primer pairs, rbcL _ For and rbcL _ Rev. OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% TritonX-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by combining:

8.4 μ L of water

2.0 μ L10 XOPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdNTPs(10 mM)

2.0 μ L primer sets rbcL _ For and rbcL _ Rev (2 or 4 μ M each)

0.5 μ LVentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

2.0 μ L of Total nucleic acid isolated from leaf tissue (3.3 ng/. mu.L)

Two primer pairs rbcL _ For and rbcL _ Rev of different concentrations were used to determine the threshold of primer concentration required For efficient amplification of rbcL gene fragments. The reaction was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 76 ℃ for 10 seconds and 60 ℃ for 10 seconds. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2. mu.L of 6X loading buffer (New England BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. The 137 bp product was clearly observed for both infected and uninfected samples and matched the expected length of the OPCRar target sequence (figure 14).

Referring now to FIG. 14, shown in a gel is the amplification of a specific target sequence present in chloroplast DNA, according to one embodiment of the invention. Total nucleic acid (3.3 ng/. mu.L) isolated from leaf tissue of (i) or (ii) infected Asian species of the species Pholiopsis citricola was used as the initial template. All reactions were performed in the presence of 10% DMSO using VentR (exo-) DNA polymerase, Et SSB, and primers rbcL _ For and rbcL _ Rev. The OPCRar solution was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 76 ℃ for 10 seconds and 60 ℃ for 10 seconds. The amplified products were visualized on a 12% acrylamide gel stained with ethidium bromide. The 137 bp product was clearly observed for all positive samples and matched the expected length of the OPCRar target sequence.

Example 7: method for multiplex amplification of target sequences and positive controls by OPCRar

To confirm that OPCRar is able to amplify more specific target sequences, we amplified nucleic acids extracted from plants tissues infected with liberibacter citreus asian species using OPCRar primer pairs hyvl _ For/hyvl _ Rev and rbcL _ For/rbcL _ Rev. These primer sets produced 139 and 137 bp products, respectively. OPCRar buffer (10X) containing 400mM Tris-HCl (pH8.4), 10 mM ammonium sulfate, 100 mM potassium chloride, and 0.25% Triton X-100 was prepared in advance. Prepare 20 μ L of OPCRar solution by combining:

6.4 μ L of water

2.0 μ L of 10x OPCRar buffer

3.0μLDMSO

0.4 μ L Potassium chloride (2M)

0.5. mu.L of magnesium chloride (100 mM)

0.5. mu.L dithiothreitol (100 mM)

0.5μLdN TPs(10 mM)

2.0 μ L primer sets rbcL _ For and rbcL _ Rev (2 or 3 μ M each)

2.0 μ primer sets hyg _ For and hyg _ Rev (8 μ M each)

0.5. mu.L of VentR (exo-) DNA polymerase (2U/. mu.L)

0.2 μ LEt SSB, extremely thermostable Single-chain binding protein (500 μ g/mL)

2.0 μ L of Total nucleic acid isolated from leaf tissue (3.3 ng/. mu.L)

Two different concentrations of primer pairs rbcL _ For and rbcL _ Rev were used to determine the threshold primer concentration required For efficient amplification of rbcL gene fragments in the presence of 800 nM primers specific For hyvl gene fragments. The reaction was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times with shaking between 76 ℃ for 10 seconds and 60 ℃ for 10 seconds. After completion of the reaction, 5. mu.L of OPCRar product was mixed with 2. mu.L of 6X loading buffer (New England BioLabs) and 1. mu.L of formamide, electrophoresed on a 12% acrylamide gel and visualized with ethidium bromide. 139 bp and 137 bp products were clearly observed, which matched the expected length of the OPCRar target sequence. For clarity, OPCRar products generated using only two primer pairs (examples 3 and 6) were run with multiplex reactions (fig. 15).

Referring now to FIG. 15, presented is a gel showing the products of multiplex amplification of two target sequences according to one embodiment of the invention. Total nucleic acid (3.3 ng/. mu.L) isolated from Asian infected leaf tissue of the species Pholiopsis citricola was used as the initial template. All reactions were performed in the presence of 10% DMSO using the indicated concentrations of VentR (exo-) DNA polymerase, Et SSB, and primer sets hyvl _ For/hyvl _ Rev and rbcL _ For/rbcL _ Rev. The multiplex OPCRar solution was heated at 85 ℃ for 2 minutes to denature the template and then cycled 40 times, shaking between 76 ℃ for 10 seconds and 60 ℃ for 10 seconds. Multiplex products were visualized on 12% acrylamide gels stained with ethidium bromide. When compared to OPCRar products generated from the primer set alone (see fig. 11 and 15), both 139 and 137 bp products were clearly observed.

Referring now to FIG. 16, shown is the amplification product from a reaction in the presence of Et SSB according to one embodiment of the present invention. Using the systems and methods of the invention, SSB increases amplification efficiency at lower melting temperatures. We investigated the use of the extremely thermostable single-chain binding protein "ET SSB", and demonstrated that it contributes to the performance of OPCRar at some melting temperatures. Using purified leaf sample DNA containing the HLB disease gene, OPCRar reactions were performed on the HLB pathogen target. The template is DNA purified from citrus leaves of a tree infected with liberibacter citreus. The primers were hyvl _ For/and hyvl _ Rev. The thermal cycler conditions were: initial melting at 85 ℃ for 2 minutes, followed by 40 cycles of denaturation at 76 ℃ or 74 ℃ for 10 seconds and annealing at 60 ℃ for 10 seconds. The results indicate that the presence of Et SSB resulted in amplification of the liberibacter citreum target at all temperature states assayed. Comparative OPCRar experiments were performed in duplicate in the presence of ET SSB egg changes and in the absence of ET SSB. A standard PCR thermal cycler with precise temperature control was used to allow evaluation of amplification performance in the presence and absence of SSB. The results indicate that we see amplified HLB at 74 ℃ in the presence of ET SSB, and that in the absence of ET SSB, amplified HLB is not visible.

Referring now to FIG. 17, gel electrophoresis of an OPCRar reaction using hyg _ For and hyg _ Rev primers and purified M.citricola DNA isolated from the leaves of infected trees is shown, wherein the reaction does not include the temperature-variable parameters involved in a typical PCR thermocycler (FIG. 17A) or the temperature-variable time to cycling temperature (FIG. 17B), according to one embodiment of the present invention. As used herein, Ramping refers to heating (e.g., the heating process that raises the amplification reaction temperature from the annealing temperature to the denaturation temperature in each thermal cycle is referred to as Ramping up, or cooling from the denaturation temperature to the annealing temperature is referred to as cooling down.) all conventional PCR cyclers and methods have a controlled heating design, where the temperature rise is approximately >3 degrees/second, and the cooling down rate is approximately >1 degrees/second. the Ramping time is not included in a typical cycle profile (profile), the instrument does not begin counting the duration until the desired temperature is reached during the denaturation, annealing, and extension phases. for example, the denaturation time is at 90 degrees for 10 seconds, the instrument will not begin counting the 10 seconds after 90 degrees, in contrast, the systems and methods of the present invention provide a low cost heater device that is designed to be, for example, at 80 degrees for 10 seconds, meaning that the time is counted as the heating process begins (rather than waiting until the desired denaturation temperature is reached). FIG. 17A: we examined OPCRar amplification of HLB disease (Citrus greening disease pathogen, Huanglongbing bang) targets in a low cost thermal heater (without active cooling and precise temperature control, > +2 degree fluctuations, (see, e.g., the system and apparatus disclosed in 61/477,357.) A20 μ L OPCRar reaction was performed in a micro-heater reaction chamber under microprocessor control in an apparatus developed by Mesa Tech International, Inc. (MTI apparatus) or in a conventional PCR thermocycler (PCR thermocycler) such that the 10 second residence time for each temperature segment of the program started immediately after the instruction to change temperature was executed by the microprocessor (i.e., temperature Change time map 17A.) is different from the 10 second residence time for each temperature segment of the program started immediately after the target temperature was detected by the temperature sensor located at the reaction orifice (i.e., temperature Change time map 17B.) PCR melting temperature cycler conditions are as follows: 85 ℃ for 2 minutes initially Followed by 40 cycles of 80 ℃ for 10 seconds and 60 ℃ for 10 seconds. MTI equipment conditions were as follows: initial melting: 40 cycles of 85 ℃ for 2 minutes, 82 ℃ for 10 seconds and 59 ℃ for 20 seconds.

FIG. 17A: first, OPCRar amplification without separate temperature ramping (20 μ l reaction). From the left: a 50bp standard DNA size ladder-shaped band; second and third lanes: OPCRar amplification reactions in a standard PCR thermocycler with temperature ramping step and precise temperature control (in duplicate, lanes 2 and 3). Initial melting: at 85 ℃ for 2 minutes, 40 cycles between denaturation at 80 ℃ for 10 seconds and annealing at 60 ℃ for 10 seconds. Lanes 4 and 5: OPCRar amplification in a low cost thermal engine without active cooling and/or separate temperature swing control. Compared with the temperature changing stage without any denaturation or annealing temperature, the temperature of the heat engine is changed from the ambient temperature (25 ℃) to 80 ℃ for denaturation or is reduced to 60 ℃ for annealing. HLB OPCRar was performed in a 20 μ L reaction using purified plant DNA containing the HLB disease target sequence. We used a PCR thermal cycler for positive control for MTI device testing. PCR thermocycler conditions were as follows: the conditions of the MTI equipment thermal cycler without temperature change are as follows: initial melting: at 85 ℃ for 2 minutes, cycling was performed between denaturation at 82 ℃ for 10 seconds and annealing at 59 ℃ for 20 seconds. The cycle was repeated 40 times. MTI plant thermocyclers for the ramp-up phase are the same except that there is a ramp-up phase of <10 seconds and a ramp-down phase of <20 seconds. The data indicate that HLB OPCRar can still amplify HLB amplicons even in the absence of the warm-up and cool-down phases. Comparison of 17A and 17B reveals a significant increase in amplification product yield when the temperature ramp time is set.

Referring now to fig. 18, shown in the gel are: the present invention according to one embodiment comprises two primer sets in the MTI thermistor-based low-cost amplification apparatus as described above: 1) primers hyg _ For and hyg _ Rev specific to the citrus yellow dragon pathogen DNA target and 2) primers rbcL (ribulose-1, 5-bisphosphate carboxylase oxygenase) For and rbcL _ Rev specific to the citrus housekeeping gene rbcL. The reaction products resulting from the PCR amplification and carried out in a low cost heater without temperature swing (e.g. 5 degrees/second in a PCR setup) and without precise temperature control were electrophoresed on a gel. We tested the RbCL internal positive control with the HLB primers in a PCR thermocycler and MTI device (with or without a temperature-shift program). We performed a 40 uL reaction using a purified HLB sample. PCR thermocycler conditions were as follows: initial melting: 2 minutes at 85 ℃ and cycles between 10 seconds of denaturation at 80 ℃ and 10 seconds of annealing at 60 ℃. MTI device amplification conditions were as follows: initial melting: 90 ℃ for 2 minutes, between denaturation at 82 ℃ for 10 seconds and annealing at 59 ℃ for 20 seconds. We found that it was still able to amplify the HLB and RbCL primer sequences without temperature shift. The HLB product is about 147bp and the RbCL product is about 140 bp. The MTI apparatus used for both conditions was comparable to the PCR thermal cycler amplified product. The forward primer for this reaction was ccagccttga tcgttacaaa gggcgatgct acaacatt (SEQ ID NO 9) (Tm was about 73.9C (10% DMSO, 76/60C), the reverse primer was catgttagta acagaacctt cttcaaaaaggtctaacggg taa (SEQ ID NO 10) (Tm was about 71.2C (10% DMSO, 76/60℃) the OPCRar reaction was performed with or without temperature shift time in the temperature residence time (as described for FIGS. 16 and 17), as indicated. 40. mu.L reaction was performed using purified citrus leaf DNA from a Citrus flavedo-infected tree. the PCR thermocycler control conditions were initial melting for 2 minutes at 85 ℃ and 40 cycles at 10 seconds at 80 ℃ and 10 seconds at 60 ℃ and 40 cycles at 10 seconds at 59 ℃ the MTI device amplification conditions were initial melting for 2 minutes at 90 ℃, 10 seconds at 82 ℃ and 20 seconds at 59 ℃ the results revealed that when temperature shift time was not included in the residence time calculation, the MTI device is capable of amplifying both liberibacter citreus and rbcL sequences. The product of the xanthomonas citri is about 147bp (HLB), and the product of rbcL (RbCL) is about 140 bp.

All Primer melting temperatures (Tm) were calculated using IDT OligoAnalyzer 3.1(Integrated DNA Technologies, inc., Coralville, IA), using Primer3 Tm calculation software where salt, dNTP, Mg, Primer concentration parameters were taken into account, using the following parameters:

concentration of oligonucleotide: 0.25 μ M; na (Na)⁺Concentration: 50 mM; mg (magnesium)⁺⁺Concentration =2.5 mM; dNTP concentration =0.25 μ M. The symbol "a" refers to adenine, "g" refers to guanine, "c" refers to cytosine, "t" refers to thymine, "u" refers to uracil, "r" refers to purine, "y" refers to pyrimidine, "m" refers to amino, "k" refers to ketone, "n" refers to either a or g or c or t/u, unknown, or otherwise.

(SEQ ID NO 1)

Registration number: CY087034

Type (2): viral RNA

Length: 1010

An organism: influenza A virus (H1N1)

Other information: matrix protein 2(M2) and matrix protein 1(M1) genes

(SEQ ID NO 2)

Type (2): forward primer

Name: FP3

Length: 46

Tm: average 75 deg.C

(SEQ ID NO 3)

Type (2): reverse primer

Name: RP3

Length: 40

Tm: average 77.8 deg.C

(SEQ ID NO 4)

Type (2): reverse primer

Name: RP4

Length: 46

Tm: average 74.7 deg.C

(SEQ ID NO 5)

Type (2): forward primer

Name: UniAfCDC

Length: 22

Tm: average 65.0 deg.C

(SEQ ID NO 6)

Type (2): reverse primer

Name: UniArCDC

Length: 24

Tm: average 66.6 deg.C

(SEQ ID NO 7)FAM-tgcagtcctc gctcactggg cacg-BHQ

Type (2): TaqMan Probe

Name: UniApCDC

Length: 24

Tm：73.4℃

(SEQ ID NO 8)

Registration number: AB505957

Type (2): chloroplast DNA

Length: 1326

An organism: sweet orange (Citrus sinensis)

Other information: rbcL, ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit

(SEQ ID NO 9)

Type (2): forward primer

Name: rbcL _ For

Length: 38

Tm：73.9℃

(SEQ ID NO 10)

Type (2): reverse primer

Name: rbcL _ Rev

Length: 43

Tm：71.2℃

(SEQ ID NO 11)

Registration number: from EU523377

Type (2): bacterial 1 DNA

Length: 890

An organism: arthrobacter asiaticus (Candidatus Liberibacter asiaticus)

Other information: elongation factor Ts

(SEQ ID NO 12)

Type (2): forward primer

Name: NBEU523377-F-57

Length: 57

Tm：75.8℃

(SEQ ID NO 13) [ Biotin-5 ] tcttcgtatc ttcatgcttc tccttctgag ggtttaggatcgattggtgt tcttgta

Type (2): biotinylated forward primer

Name: EU523377-F-57

Length: 57

Tm：75.8 ℃

(SEQ ID NO 14)

Type (2): forward primer

Name: HLBForSh

Length: 47

Tm：75.6℃

(SEQ ID NO 1 5)

Type (2): reverse primer

Name: EU523377-R-56

Length: 56

Tm：75.8℃

(SEQ ID NO 16)

Type (2): reverse primer

Name: HLBRevSh

Length: 49

Tm：75.5℃

(SEQ ID NO 17)

Target: 16S ribosomal RNA of citrus yellow dragon germ Asian species

Type (2): forward primer (underlined), containing the 5' detection sequence

Name: HLBas-P2

Length: 39

Tm：62.7℃

(SEQ ID NO 1 8)

Target: 16S ribosomal RNA of citrus yellow dragon germ Asian species

Type (2): reverse primer (underlined) containing the 5' T7 promoter

Name: HLBr-P1

Length: 56

Tm：64.5℃

(SEQ ID NO 19)

Type (2): forward primer

Name: hyvl _ For

Length: 45

Tm：72.2℃

(SEQ ID NO 20)

Type (2): reverse primer

Name: hyg _ Rev

Length: 51

Tm：70.9℃

While the invention has been described in detail with particular reference to the described embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art, and it is intended to cover all such modifications and equivalents. The entire disclosures of all patents and publications cited above are incorporated by reference.

Claims (46)

1. A method of amplifying a template of a nucleic acid target sequence contained in a sample, the method comprising:

contacting the sample with an amplification reaction mixture comprising a primer complementary to a template of the nucleic acid target sequence;

oscillating the temperature of the reaction between an upper temperature limit and a lower temperature limit, wherein the temperature changes by no more than 20 ℃ during a plurality of temperature cycles; and

amplifying a template of the nucleic acid target sequence,

wherein the amplification reaction mixture comprises a primer pair that binds to opposite strands of a template of the nucleic acid,

wherein the amplification reaction mixture comprises a nucleic acid destabilizing agent comprising DMSO, formamide, and/or betaine at a concentration of 8-15 vol%.

2. The method of claim 1, wherein the temperature changes by no more than 15 ℃ during the plurality of temperature cycles.

3. The method of claim 1, wherein the temperature changes by no more than 10 ℃ during the plurality of temperature cycles.

4. The method of claim 1, wherein the temperature changes by no more than 5 ℃ during the plurality of temperature cycles.

5. The method of claim 1, wherein once said upper temperature or said lower temperature is reached, said temperature is maintained within temperature fluctuations for a period of time.

6. The method of claim 1, wherein the temperature is changed to another temperature once an upper or lower temperature within the temperature range is reached.

7. The method of claim 1, wherein the lower temperature is not less than 50 ℃.

8. The process of claim 1, wherein the upper temperature limit is no greater than 85 ℃.

9. The method of claim 1, wherein the template for the nucleic acid target sequence is single-stranded DNA or RNA.

10. The method of claim 1, wherein the template for the nucleic acid target sequence is double-stranded DNA or RNA.

11. The method of claim 1, wherein the template of the nucleic acid target sequence is RNA.

12. The method of claim 1, wherein the template of the nucleic acid target sequence is DNA.

13. The method of claim 1, wherein the target nucleic acid can be less than 1000bp in length.

14. The method of claim 1, wherein the target nucleic acid can be less than 250bp in length.

15. The method of claim 1, wherein the target nucleic acid can be less than 150bp in length.

16. The method of claim 1, wherein the target nucleic acid can be less than 100bp in length.

17. The method of claim 1, wherein the primer pair has a length and GC content such that the melting temperature is greater than or equal to 65 ℃.

18. The method of claim 1, wherein the primer pair has a length and a GC content such that the melting temperature is 70 ℃ or higher.

19. The method of claim 1, wherein the primer pair has a length of 35-70 base pairs.

20. The method of claim 1, wherein the melting temperature of each primer of the primer pair is 70-80 ℃.

21. The method of claim 1, wherein the primer pair comprises a forward primer and a reverse primer, each of the forward primer and the reverse primer having a length of 40-47 base pairs.

22. A method of amplifying a template of a nucleic acid target sequence contained in a sample, the method comprising:

contacting the sample with an amplification reaction mixture comprising

A primer or primer pair having a length of 35-70 base pairs and being complementary to a template of the nucleic acid target sequence, and wherein each primer of the primer pair has a melting temperature of 70-80 ℃;

DMSO, formamide and/or betaine at a concentration of 8-15% by volume;

a monovalent cation;

a divalent cation;

dNTPs; and

a DNA polymerase;

oscillating the temperature of the reaction between an upper temperature limit and a lower temperature limit, wherein the temperature changes by no more than 20 ℃ during a plurality of temperature cycles; and

amplifying the template of the nucleic acid target sequence.

23. The method of claim 22, wherein the divalent cation is a salt selected from the group consisting of: magnesium, manganese, copper, zinc, or any combination thereof.

24. The method of claim 22, wherein said monovalent cation is a salt selected from the group consisting of: sodium, potassium, lithium, rubidium, cesium, ammonium, or any combination thereof.

25. The method of claim 1, wherein the amplification reaction comprises a DNA polymerase.

26. The method of claim 22 or 25, wherein the DNA polymerase is a thermostable DNA polymerase.

27. The method of claim 22 or 25, wherein the DNA polymerase is selected from the group consisting of: TAQ DNA polymerase, VentR DNA polymerase and deep VentR DNA polymerase.

28. The method of claim 22 or 25, wherein the DNA polymerase comprises strand displacement activity.

29. The method of claim 22 or 25, wherein the DNA polymerase does not have 3 '- > 5' exonuclease activity.

30. The method of claim 11, wherein the amplification reaction mixture comprises a reverse transcriptase and a DNA polymerase.

31. The method of claim 30, wherein the reverse transcriptase is a thermostable reverse transcriptase.

32. The method of claim 30, wherein the reverse transcriptase is selected from the group consisting of: AMV-RT, superscriptII reverse transcriptase, superscriptIII reverse transcriptase or MMLV-RT.

33. The method of claim 1 or 22, wherein the amplification reaction mixture comprises a single-chain binding protein.

34. The method of claim 33, wherein said single chain binding protein is a thermostable single chain binding protein.

35. The method of claim 33, wherein the single chain binding protein is a non-thermostable single chain binding protein.

36. The method of claim 1 or 22, wherein the sample is not alcohol-free.

37. The method of claim 1 or 22, wherein the sample is not salt-free.

38. The method of claim 1, wherein the amplification reaction mixture comprises:

single-or double-stranded nucleic acid destabilizing agents;

a monovalent cation;

a divalent cation;

dNTPs; and

a DNA polymerase buffered at a pH that supports activity.

39. The method of claim 38, wherein the amplification reaction mixture further comprises a single-chain binding protein.

40. The method of claim 38, wherein the destabilizing agent is dimethyl sulfoxide (DMSO) or formamide.

41. The method of claim 38, wherein the divalent cation is a salt selected from the group consisting of: magnesium, manganese, copper, zinc or any combination thereof.

42. The method of claim 38, wherein said monovalent cation is a salt selected from the group consisting of: sodium, potassium, lithium, rubidium, cesium, ammonium, or any combination thereof.

43. The method of claim 38, wherein the DNA polymerase is a thermostable DNA polymerase.

44. The method of claim 38, wherein the DNA polymerase is selected from the group consisting of: TAQ DNA polymerase, VentR DNA polymerase and deep VentR DNA polymerase.

45. The method of claim 38, wherein the DNA polymerase comprises strand displacement activity.

46. The method of claim 38, wherein the DNA polymerase does not have 3 '- > 5' exonuclease activity.