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WO2025151807A1 - Extraction d'échantillon de lysat brut pour pcr numérique - Google Patents

Extraction d'échantillon de lysat brut pour pcr numérique

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Publication number
WO2025151807A1
WO2025151807A1 PCT/US2025/011220 US2025011220W WO2025151807A1 WO 2025151807 A1 WO2025151807 A1 WO 2025151807A1 US 2025011220 W US2025011220 W US 2025011220W WO 2025151807 A1 WO2025151807 A1 WO 2025151807A1
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WO
WIPO (PCT)
Prior art keywords
sample
nucleic acid
crude lysate
crude
pcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/011220
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English (en)
Inventor
Wei Wei
Jennifer Berkman
David Joun
Chengjing LIU
Junko Stevens
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Life Technologies Corp
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Life Technologies Corp
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Publication date
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Publication of WO2025151807A1 publication Critical patent/WO2025151807A1/fr
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Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present disclosure is in the field of molecular biology and genetic analysis.
  • the present disclosure relates to methods, compositions, polymerase chain reaction (PCR) systems, and kits useful for the extraction, amplification, and detection of nucleic acids from a crude lysate sample. More specifically, methods, compositions, PCR systems, and kits are provided for the amplification, detection, and/or quantitation of nucleic acids from a crude lysate sample by digital polymerase chain reaction (dPCR).
  • dPCR digital polymerase chain reaction
  • amplification of a particular nucleic acid sequence is essential to allow detection of the particular nucleic acid sequence in, or isolation from, a sample in which nucleic acid (having the particular nucleic acid sequence) is present in very low amounts.
  • Plant diseases caused by plant pathogens cost the global economy $220 billion annually (see, for example, Savary et al., “[t]he global burden of pathogens and pests on major food crops,” Nat. Ecol. Evol. Vol. 3, pp. 430-439, 2019). Early detection of plant diseases is a crucial factor to prevent or limit the spread of infection that could cause significant economic loss. Traditional methods of detection, such as symptom development or pathogen culturing, are time-consuming and may be too late to stop crop loss or disease spread. [0005] One such plant pathogen is Xylella fastidiosa ( “X.
  • fastidiosa a pathogenic bacterium affecting over 600 plant species, including plants of major socioeconomic interest, such as, for example, olive trees and grapevines (see, for example, EFSA, “[u]pdate of the Xylella spp. host plant database,” EFSA J. 16, 2018).
  • X. fastidiosa causes severe crop diseases including Pierce’s disease of grapevines (see, for example, Pierce, “[t]he California vine disease,” U.S. Dep. Agric. Div. Veg. Pathol. Bull., Vol. 2, p. 222, 1892).
  • fastidiosa infects the xylem vessels of plants (see, for example, Almeida et al., “[h]ow do plant diseases caused by Xylella fastidiosa emerge?,” Plant Dis., Vol. 99, pp. 1457-1467, 2015).
  • X. fastidiosa is transmitted to plants via insects, making it difficult to control the spread of disease.
  • Visible disease symptoms due to X. fastidiosa can take several months to develop, and the pathogen itself is challenging to culture in a laboratory, making early detection of the pathogen difficult.
  • a method that can detect low levels of Xylella fastidiosa infection during early stages of infection is vital. Accordingly, molecular detection methods are preferred for disease identification and surveillance.
  • PCR Polymerase chain reaction
  • qPCR quantitative PCR
  • FIG. 5 shows a conventional sample preparation and amplification workflow requiring a purification step.
  • a crude sample is mixed with a lysis solution in 503, and vortexed to lyse cells of the crude sample. After vortexing, the mixture is incubated for an amount of time sufficient for lysing.
  • chloroform such as phenol: chloroform: isoamyl alcohol
  • chloroform such as phenol: chloroform: isoamyl alcohol
  • a precipitation solution is added to the aqueous phase to precipitate the extracted nucleic acid.
  • the mixture is incubated for a desired time, and then centrifuged to obtain a pellet of nucleic acid.
  • the supernatant is removed, and the pellet is washed with alcohol (e.g., ethanol) in 513, and centrifuged to obtain a pellet of purified nucleic acid.
  • alcohol e.g., ethanol
  • the ethanol is removed, and the pellet of purified nucleic acid is dissolved in a desired buffer or solution.
  • qPCR is performed on the purified sample where the purified sample is mixed with an amplification mixture in 530. Then, the amplification mixture including the purified sample is loaded onto a plate in 540. Thereafter, a thermocycle protocol is performed on the mixture in the plate in 550. Fluorescence is then detected from amplicons of target nucleic acids, and the results are analyzed in 560.
  • FIG. 6 is another conventional sample preparation and amplification workflow requiring a purification step.
  • a crude sample is mixed with a first lysis solution in 603, and vortexed. Then, a second lysis solution is added to the mixture in 605, and the mixture is vortexed to lyse cells of the crude sample.
  • the mixture is incubated for an amount of time sufficient for lysing in 607.
  • a precipitation solution is added to the mixture to precipitate nucleic acid from the mixture.
  • the mixture is incubated again, and then centrifuged to obtain a pellet of nucleic acid.
  • the supernatant is removed, and the pellet is washed with alcohol (e.g., ethanol) in 613, and centrifuged to obtain a pellet of purified nucleic acid.
  • alcohol e.g., ethanol
  • the ethanol is removed, and the pellet of purified nucleic acid is dissolved in a desired buffer or solution.
  • qPCR is performed on the purified sample where the purified sample is mixed with an amplification mixture in 630. Then, the amplification mixture including the purified sample is loaded onto a plate in 640. Thereafter, a thermocycle protocol is performed on the mixture in the plate in 650. Fluorescence is then detected from amplicons of target nucleic acids, and the results are analyzed in 660.
  • a conventional restriction enzyme incubation step includes exposing a sample to restriction enzymes that locate and bind to specific sequences or restriction sites on the nucleic acids present in the sample and cleave the nucleic acids into fragments with a known sequence at or near those sites.
  • restriction enzymes can be costly.
  • FIGS. 7 shows a conventional sample preparation and amplification workflow using a restriction enzyme.
  • a crude sample is mixed with a restriction enzyme in 702, and then the mixture of the crude sample and restriction enzyme is incubated for an amount of time sufficient for extraction in 707. For instance, the mixture can be incubated at approximately 35°C for about 30 minutes.
  • qPCR is performed on the purified sample where the purified sample is mixed with an amplification mixture in 730. Then, the amplification mixture including the purified sample is loaded onto a plate in 740. Thereafter, a thermocycle protocol is performed on the mixture in the plate in 750. Fluorescence is then detected from amplicons of target nucleic acids, and the results are analyzed in 760.
  • PCR polymerase chain reaction
  • kits for the extraction, amplification, detection, and/or quantitation of nucleic acids from a biological sample by digital polymerase chain reaction (dPCR).
  • the biological sample is a crude lysate sample.
  • a first embodiment provides a method of performing a digital polymerase chain reaction (dPCR), the method comprising performing an amplification reaction on a reaction mixture including a crude lysate sample to generate amplicons of a target nucleic acid of a pathogen, wherein the crude biological sample includes biological tissue; and detecting fluorescence from a probe hybridized to the amplicons.
  • dPCR digital polymerase chain reaction
  • the method further comprises preparing the crude lysate sample by homogenizing a crude biological sample having the biological tissue, and mixing the homogenized crude biological sample with an extraction buffer.
  • the method is performed without incubating the crude lysate sample with a restriction enzyme.
  • the extraction buffer is an aqueous extraction buffer.
  • the extraction buffer is a lysis buffer.
  • the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.
  • the preparing of the crude lysate sample further comprises centrifuging the mixture of the homogenized crude biological sample and the extraction buffer to form a supernatant, and collecting the supernatant to generate the crude lysate sample.
  • the reaction mixture is an unpurified reaction mixture.
  • the crude lysate sample is an unpurified lysate sample.
  • the preparing of the crude lysate sample and the performing of the amplification reaction are performed without extracting the crude biological sample or the crude lysate sample with a chloroform-based compound and/or a phenol -based compound.
  • the performing of the amplification reaction includes preparing the reaction mixture including the crude lysate sample, and loading the reaction mixture including the crude lysate sample onto a microfluidic array plate.
  • the loading of the reaction mixture includes transferring a portion of the reaction mixture including the crude lysate sample into a plurality of microchambers of the microfluidic array plate, and the performing of the amplification reaction includes thermocycling the reaction mixture in the plurality of microchambers.
  • the microfluidic array plate is a single plate.
  • the method further comprises quantifying an amount of the target nucleic acid based on the fluorescence detected.
  • the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.
  • the presence of the pathogen is determined before the subject is symptomatic of an infection by the pathogen.
  • the method further comprises preparing the crude lysate sample by homogenizing a crude biological sample including the biological tissue, and mixing the homogenized crude biological sample with an extraction buffer.
  • the preparing of the crude lysate sample and the performing of the amplification reaction are performed without extracting the crude biological sample or the crude lysate sample with a chloroform-based compound and/or a phenol -based compound.
  • the target nucleic acid is at least one selected from viral nucleic acid, bacterial nucleic acid, total bacterial nucleic acid, genomic nucleic acid, and fungal nucleic acid.
  • the probe emits fluorescence that is detectable when a reporter dye of the probe is cleaved from the amplicons, and an amount of the target nucleic acid is quantifiable based on the fluorescence detected.
  • the reaction mixture further includes a reverse transcriptase.
  • the target nucleic acid belongs to a pathogen infecting a host or subject.
  • the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.
  • the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.
  • the crude biological sample is prepared by homogenizing or grinding a crude biological sample including the biological tissue, and mixing the homogenized biological sample with an extraction buffer.
  • the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.
  • the extraction buffer is a lysis buffer.
  • the extraction buffer is an aqueous extraction buffer.
  • the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.
  • the reaction mixture is an unpurified reaction mixture, and/or the crude lysate sample is an unpurified biological sample.
  • the crude lysate sample is prepared without extracting the crude biological sample or the crude lysate sample with a chloroform -based compound and/or a phenol-based compound.
  • the amplification reaction is performed without extracting the crude lysate sample with a chloroform-based compound and/or a phenol -based compound.
  • the target nucleic acid belongs to a pathogen infecting a host or subject.
  • the crude lysate sample is prepared by homogenizing or grinding a crude biological sample including the biological tissue, and mixing the homogenized crude biological sample with the extraction buffer.
  • the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.
  • the extraction buffer is a lysis buffer.
  • the extraction buffer is an aqueous extraction buffer.
  • the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.
  • the reaction mixture is an unpurified reaction mixture.
  • the crude lysate sample is an unpurified biological sample.
  • the crude lysate sample is prepared without extracting the crude biological sample or the crude lysate sample with a chloroform -based compound and/or a phenol-based compound.
  • the target nucleic acid in the crude lysate sample is amplified without extracting the crude biological sample or the crude lysate sample with a chloroformbased compound and/or a phenol-based compound.
  • the target nucleic acid is amplified on a microfluidic array plate of a digital PCR instrument.
  • the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored. [00108] In the fourth embodiment, the microfluidic array plate is a single plate.
  • the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.
  • the crude lysate sample is a biological tissue sample in the form of at least one selected from plasma, serum, biological fluids, semen, saliva, whole blood, feces, milk, organ and hair.
  • the crude lysate sample is an environmental sample in the form of at least one selected from a water sample, an air sample, a plant sample, a fungal sample, and a soil sample.
  • the target nucleic acid is at least one selected from viral nucleic acid, bacterial nucleic acid, total bacterial nucleic acid, genomic nucleic acid, and fungal nucleic acid.
  • FIGS. 1-8 represent non-limiting, example embodiments as described herein.
  • FIG. 1A shows variability charts for a first 4-week-infected grapevine tissue sample as a crude lysate sample after dPCR assay without a conventional restriction enzyme incubation step prior to dPCR (“Omins”) and as a purified sample with a conventional restriction enzyme incubation step prior to dPCR (“30mins 35°C”).
  • FIG. IB shows variability charts for a second 4-week-infected grapevine tissue sample as a crude lysate sample after dPCR assay without a conventional restriction enzyme incubation step prior to dPCR (“Omins”) and as a purified sample with a conventional restriction enzyme incubation step prior to dPCR (“30mins 35°C”).
  • FIG. 1C shows variability charts for a first 8-week-infected grapevine tissue sample as a crude lysate sample after dPCR assay without a conventional restriction enzyme incubation step prior to dPCR (“Omins”) and as a purified sample with a conventional restriction enzyme incubation step prior to dPCR (“30mins 35°C”).
  • FIG. ID shows variability charts for a second 8-week-infected grapevine tissue sample as a crude lysate sample after dPCR assay without a conventional restriction enzyme incubation step prior to dPCR (“Omins”) and as a purified sample with a conventional restriction enzyme incubation step prior to dPCR (“30mins 35°C”).
  • FIG. 2A shows copy number detection levels in the dPCR assay of FIG. 1 A for the purified sample.
  • FIG. 2B shows copy number detection levels in the dPCR assay of FIG. 1 A for the crude lysate sample.
  • FIG. 2C shows copy number detection levels in the dPCR assay of FIG. IB for the purified sample.
  • FIG. 2D shows copy number detection levels in the dPCR assay of FIG. IB for the crude lysate sample.
  • FIG.3A shows copy number detection levels in the dPCR assay of FIG. 1C for the purified sample.
  • FIG. 3B shows copy number detection levels in the dPCR assay of FIG. 1C for the crude lysate sample.
  • FIG. 3C shows copy number detection levels in the dPCR assay of FIG. ID for the purified sample.
  • FIG. 3D shows copy number detection levels in the dPCR assay of FIG. ID for the crude lysate sample.
  • FIG. 4 shows correlation data between detected quantities of the target nucleic acid by dPCR and qPCR with and without the conventional restriction enzyme incubation step.
  • FIGS. 5, 6 and 7 show conventional sample preparation and amplification workflows.
  • FIG. 8 shows a simplified, streamlined sample preparation and amplification workflow according to embodiments.
  • first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of embodiments.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items.
  • PCR polymerase chain reaction
  • kits for the extraction, amplification, detection, and/or quantitation of nucleic acids from a biological sample by digital polymerase chain reaction (dPCR).
  • the biological sample is a crude lysate.
  • dPCR Digital polymerase chain reaction
  • qPCR real-time or quantitative PCR
  • qPCR measures PCR amplification against a reference as it occurs. Data are collected in real time during the exponential (log) phase of PCR. Bulk reaction fluorescence is measured at each cycle until a plateau phase is reached. Advantages of qPCR include improved tolerance to some PCR inhibitors, broad acceptance with well-established protocols and assays, increased dynamic range of detection, detection capability down to a two-fold change, higher sample throughput with lower cost, and providing a permanent record of amplicon amplification by collecting data in the exponential phase of PCR.
  • dPCR provides an absolute measurement by counting the number of the target of interest via single-molecule amplification across a large number of PCR replicates. Steps of dPCR include distribution reaction, amplification, and counting at an endpoint. dPCR includes a run at a limiting dilution to ensure at least one reaction does not contain any target nucleic acid(s).
  • dPCR a quantitative data output without reliance on references or standards for conversion of data points, the capability to analyze rare targets against wild-type or a non-target background, provision of a linear response to the number of copies present to allow for small fold-change differences to be detected, and single-molecule resolution interrogation that enables identification and quantification of molecules containing multiple targets, such as for example, phased targets or engineered plasmids.
  • the reaction mixture is partitioned into many small reaction volumes (i.e., partitions), so that the target nucleic acid is in some, but not all, of the reaction volumes or partitions.
  • the reaction volumes are subjected to thermal cycling, and the proportion of “positive” partitions that generate a signal, usually a fluorescence signal, indicative of the presence of the target, is determined.
  • Quantitation is based on application of Poisson statistics, using the number of negative/non-reactive reaction volumes and assuming a Poisson distribution to establish the number of initial copies that were distributed across all the reaction volumes.
  • dPCR provides absolute quantification
  • dPCR has a high inhibitor tolerance (i.e., detection of pathogens such as Xylella fastidiosa (bacteria) DNA from insect vectors)
  • dPCR has a low limit of detection of microbes in samples such as plant, human, animal, environmental samples
  • dPCR permits analysis of rare or limited field samples.
  • the small reaction size required for dPCR systems helps mitigate any inhibition when performing qPCR that utilizes larger sample volumes.
  • a method surprisingly detects infections at an earlier stage than conventional PCR methods.
  • the method detects infection prior to any symptoms of the infection. For instance, the method detects X. fastidiosa from asymptomatic infected grapevines at 4 weeks post infection, while the grapevines are still asymptomatic of the infection, using crude lysate samples which eliminates the steps needed for full DNA extraction and/or purification.
  • embodiments are not limited thereto.
  • the methods, systems, compositions and kits according to embodiments may be used to detect pathogens (a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, or a disease-causing parasite) other than plant pathogenic bacteria.
  • pathogens a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, or a disease-causing parasite
  • the method is a streamlined method that is free of, avoids, or eliminates a conventional restriction enzyme incubation step or a purification step that is used in conventional PCR.
  • restriction enzymes can be used to digest genomic DNA from a host because genomic DNA is too large to pass through microchannels of a dPCR plate and/or clog microchambers, thus inhibiting PCR. As indicated above, the restriction enzymes are costly.
  • FIG. 8 shows a simplified, streamlined sample preparation and amplification workflow according to embodiments.
  • a crude lysate sample is prepared by homogenizing or grinding the crude sample, and mixing the crude sample with an extraction buffer in 810.
  • dPCR is performed on the crude lysate sample where the crude lysate sample is mixed with an amplification mixture in 830. Then, the amplification mixture including the crude lysate sample is loaded onto a microfluidic array plate in 840. Thereafter, a thermocycle protocol is performed on the mixture in the microfluidic array plate in 850. Fluorescence is then detected from amplicons of target nucleic acids, and the results are analyzed in 860.
  • the method is a dPCR workflow process for preparing and utilizing a crude lysate sample.
  • the crude lysate sample is a biological tissue sample in the form of at least one selected from plasma, serum, biological fluids, semen, saliva, whole blood, feces, milk, organ and hair.
  • the crude lysate sample is an environmental sample in the form of at least one selected from a water sample, an air sample, a plant sample, a fungal sample, and a soil sample.
  • the microfluidic array plate is a QuantStudioTM Absolute QTM MAP 16 plate (Thermo Fisher Scientific).
  • the preparing of the crude lysate sample includes homogenizing or grinding sample tissue in an aqueous extraction buffer.
  • the aqueous extraction buffer includes both (i) guanidinium thiocyanate (GITC), ethylenediaminetetraacetic acid (EDTA), and sodium lauroyl sarcosinate (sarcosine) (“GES”), and (ii) polyvinylpyrrolidone (PVP), referred herein as a GES-PVP extraction buffer.
  • GITC guanidinium thiocyanate
  • EDTA ethylenediaminetetraacetic acid
  • GES sodium lauroyl sarcosinate
  • PVP polyvinylpyrrolidone
  • the GES-PVP extraction buffer includes about 60% (w/v) of GITC, about 20% of EDTA, about 1% of sarcosine, and the remainder of PVP.
  • Embodiments of the aqueous extraction buffer are not limited to the GES-PVP extraction buffer. That is, other extraction buffers known in the art may be used.
  • setting up digestion by the preparing of the crude lysate sample and thereafter directly conducting dPCR by performing a dPCR amplification reaction simplifies and streamlines the testing procedure into a “one-step” process, as shown in FIG. 8.
  • the streamlined method has a lower cost than conventional molecular analysis of biological tissue and/or detection of pathogens by forgoing labor intensive and lengthy workflows and/or not requiring expensive reagents (such as the purification workflows and restriction extraction incubation workflow shown in FIGS. 5 -7).
  • the streamlined method is significantly faster than performing a complete DNA extraction or restriction enzyme incubation followed by qPCR; and the method facilitates high-throughput testing.
  • the methods disclosed herein are useful in a wide range of applications and assays that involve detecting, quantitating, and/or characterizing target nucleic acids.
  • the methods, compositions, reaction systems, and kits are used on a crude sample.
  • the term “crude sample” refers to a biological sample that has not been subjected to an organic extraction or purification to provide a purified nucleic acid sample.
  • the biological sample is subjected to homogenizing/grinding and extraction with an aqueous extraction buffer to form the crude lysate sample.
  • the crude lysate sample is not produced by extraction using an organic solvent system, such as, for example, a phenol -chloroform extraction, or other chemical extraction.
  • the dPCR assay is a simplex dPCR. In some embodiments, the dPCR is a multiplex dPCR.
  • the term “simplex” or “simplex dPCR” as used herein refers to an assay that provides for amplification of a single product within a reaction vessel. The product is primed using a distinct primer pair. A simplex reaction may further include a labeled probe specific for the amplified product, wherein the probe is detectably labeled with detectable moiety, such as a fluorescent dye.
  • the term “multiplex” or “multiplex dPCR” as used herein refers to an assay that provides for simultaneous amplification of two or more products within the same reaction vessel.
  • a multiplex reaction may further include labeled probes specific to each product, wherein the probes are detectably labeled with different detectable moieties.
  • multiplex dPCR includes those in which: (i) a multiplicity of targets are amplified in a single sample; two or more targets in one sample or (ii) multiple samples are simultaneously amplified; two targets in two different samples within a single reaction at substantially the same time.
  • the amplification or reaction mixture includes “hot start” components or steps to further prevent, reduce or eliminate nonspecific nucleic acid synthesis.
  • hot start reactions include manual techniques, barriers, reversible polymerase inactivation, and specially-designed hairpin primers.
  • components or compounds used for hot start reactions, such as in PCR can be any of those which prevent non-specific amplification of DNA by inactivating polymerase activity at lower temperature, such as during the annealing phase, while allowing reactivation or activation of the polymerase activity at a higher temperature, such as during the extension phase.
  • Antibodies for hot start PCR can be generated or selected by various methods known in the art.
  • a commercially available antibody can be used, for example, the TaqStart Antibody (Clontech) which is effective with any Taq-derived DNA polymerase, including native, recombinant, and N-terminal deletion mutants.
  • a suitable hot start primer is a primer specially designed to have secondary structure (such as a hairpin primer) which prevents the primer from annealing until cycling temperatures cause them to denature and unfold.
  • An appropriate concentration of the compound or reagent for hot start PCR in the assembled reaction mixtures can be determined by a number of methods known in the art or, for a commercial product, suggested by the manufacturer.
  • An alternative method of hot start amplification is reversible polymerase inactivation.
  • the polymerase is reacted with an antibody or an oligonucleotide aptamer that binds to the polymerase's nucleotide binding domain, rendering the polymerase inactive.
  • an antibody or an oligonucleotide aptamer that binds to the polymerase's nucleotide binding domain, rendering the polymerase inactive.
  • a monoclonal antibody to Taq polymerase such as the anti-Taq DNA polymerase antibody available from Sigma, is introduced into the reaction mixture. Upon heating, the compound dissociates from the polymerase, restoring enzyme activity.
  • U.S. Pat. No. 5,677,152 describes a method in which the DNA polymerase is chemically modified to ensure that it only becomes active at elevated temperatures.
  • hot start components or mechanisms in addition to those described above, are also well known to those of ordinary skill in the art and will be readily selectable based on their ability to work in accordance with the present teachings.
  • methods are provided that comprise at least two different hot start mechanisms, components, or steps that are used to inhibit or substantially inhibit the polymerase activity of a nucleic acid polymerase under a first condition (such as at a lower temperature) and allow polymerase activation under a second condition (such as at a higher temperature).
  • Such hot start mechanisms include, but are not limited to those described above, including antibodies or combinations of antibodies that block DNA polymerase activity at lower temperatures, oligonucleotides that block DNA polymerase activity at lower temperatures, reversible chemical modifications of the DNA polymerase that dissociate at elevated temperatures, amino acid modifications of the DNA polymerase that provide reduced activity at lower temperatures, fusion proteins that include hyperstable DNA binding domains and topoisomerase, temperature dependent ligands that inhibit the DNA polymerase, single stranded binding proteins that sequester primers at lower temperatures, modified primers or modified dNTPs.
  • the methods also employ a hydrolysis probe for the detection of nucleic acids.
  • Hydrolysis probes take advantage of the 5' exonuclease activity of some polymerases.
  • a polymerase such as Taq polymerase, uses an upstream primer as a binding site and then extends.
  • the hydrolysis probe is then cleaved during polymerase extension at its 5' end by the 5'-exonuclease activity of the polymerase.
  • upstream and downstream are used herein in relation to the synthesis of the nascent strand that is primed by a target-specific primer.
  • a target-specific probe hybridized to a region of the target nucleic acid that is "downstream" of the region of the target nucleic acid to which the primer is hybridized is located 3 ' of the primer and will be in the path of a polymerase extending the primer in a 5' to 3' direction.
  • the TaqMan® assay (see, e g. , U.S. Patent 5,210,015, incorporated herein by reference in its entirety) is an example of a hydrolysis-probe based assay.
  • hydrolysis probes are typically labeled with a reporter on the 5' end and a quencher on the 3' end. When the reporter and quencher are fixed onto the same probe, they are forced to remain in close proximity. This proximity effectively quenches the reporter signal, even when the probe is hybridized to the target sequence.
  • the hydrolysis probes are cleaved during polymerase extension at their 5' end by the 5'-exonuclease activity of Taq.
  • hydrolysis probes are often designed with a Tm that is roughly 10°C higher than the primers in the reaction. Uses of the real-time hydrolysis probe reaction are also described in U.S. Patent Nos. 5,538,848, 6,653,473, 7,485,442 and 7,205,105, the disclosures of all of which are incorporated herein by reference in their entireties.
  • the methods disclosed herein involve the use of a TaqMan assay for nucleic acid analysis and/or detection.
  • compositions of the present disclosure can be used in methods involving TaqMan probes and/or assays.
  • assays can include, but are not limited to gene expression assays (e.g., TaqManTM Gene Expression Assays), copy number variation assays (e g., TaqManTM Copy Number Assays), genotyping assays (e.g., TaqManTM Drug Metabolism Genotyping Assays or TaqManTM SNP Genotyping Assays), miRNA assays (e.g., TaqManTM MicroRNA Assays) or RNA quantitation assays (e.g., two-step reverse transcription-polymerase chain reaction assays), and TaqManTM Low Density Array Assays.
  • gene expression assays e.g., TaqManTM Gene Expression Assays
  • copy number variation assays e.g., TaqManTM Copy Number Assays
  • genotyping assays e.g., TaqManTM Drug Metabolism Geno
  • a reaction vessel may be a well in a microtiter plate (e.g., 16-well plate, 96-well plate, 384-well plate) such as a QuantStudioTM Absolute QTM MAP 16 plate (Thermo Fisher Scientific), a TaqManTM Array plate (Applied BiosystemsTM; Thermo Fisher Scientific), a spot on a glass slide, a well in an Applied BiosystemsTM TaqManTM Array Card or Plate (Thermo Fisher Scientific) or a through-hole of an Applied BiosystemsTM TaqManTM OpenArrayTM plate (Thermo Fisher Scientific).
  • a plurality of reaction vessels may reside on the same support.
  • lab-on-a- chip-like devices available for example from Caliper and Fluidigm, can provide for reaction vessels.
  • various microfluidic approaches may be employed. It will be recognized that a variety of reaction vessels are available in the art and fall within the scope of the present teachings.
  • amplicon and “amplification product” or “amplified product” as used herein generally refer to the product of an amplification reaction.
  • An amplicon may be doublestranded or single-stranded and may include the separated component strands obtained by denaturing a double-stranded amplification product.
  • the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.
  • Exemplary methods include polymerase chain reaction (PCR), partial destruction of primer molecules (see, e.g., PCT Application Pub WO 2006/087574), ligase chain reaction (see, e g., Wu et al. Genomics 4:560-569 (1990) and Barany et al. Proc. Natl. Acad. Sci. USA 88: 189-193 (1991), QP RNA replicase systems (see, e.g., WO 1994/016108), RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; Lizardi et al. Nat. Genet.
  • PCR polymerase chain reaction
  • partial destruction of primer molecules see, e.g., PCT Application Pub WO 2006/087574
  • ligase chain reaction see, e g., Wu et al. Genomics 4:560-569 (19
  • nucleic acid amplification reaction can be performed by PCR.
  • the PCR can be endpoint PCR.
  • the PCR can be digital PCR.
  • the PCR can comprise thermal cycling. In some embodiments, the thermal cycling can be optimized for fast thermal cycling.
  • the methods for amplifying a nucleic acid by PCR comprises adding an amplification mixture to a reaction vessel; adding a crude lysate sample and a primer to the reaction vessel to form a reaction mixture; and performing PCR on the mixture including the crude lysate sample.
  • the PCR continues to occur for up to 72 hours (e.g., for up to 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours) following the addition of the amplification mixture, the crude lysate sample, and primer to the reaction vessel.
  • the methods for amplifying a target nucleic acid can be multiplex PCR amplifications in which multiple targets are simultaneously amplified.
  • the number of targets amplified can be up to as many as 2 targets, 5 targets, 10 targets 25 targets, 50 targets, 100 targets, 1000 targets, 5000 targets, and so on, including all numbers in between.
  • one of the multiplexed targets is an endogenous or an exogenous internal positive control for amplification.
  • the methods for detecting a target nucleic acid can be multiplex PCR and/or detection assays in which multiple targets are simultaneously detected.
  • the number of targets amplified and/or detected can be up to 25 targets, 10 targets, 8 targets, 6 targets, 5 targets, 4 targets, 3 targets, or 2 targets.
  • one of the multiplexed targets is an endogenous or an exogenous internal positive control for amplification and/or detection.
  • a multiplicity of targets are simultaneously amplified and detected.
  • a multiplicity of targets are amplified and detected in the same reaction vessel.
  • various TaqManTM probe reporter dye and passive dye options can be combined for multiplex PCR.
  • TaqManTM probes with FAMTM, VICTM, and ABYTM reporter dyes in combination with a PCR composition containing ROX passive reference dye may be used for a 3-plex multiplex PCR amplification and detection assay.
  • TaqManTM probes with FAMTM, VICTM, ABYTM, and JUNTM reporter dyes in combination with a PCR composition containing MUSTANG PURPLETM passive reference dye may be used for a 4-plex multiplex PCR amplification and detection assay.
  • TaqManTM probes with FAMTM, VICTM, ABYTM, JUNTM, CyTM and Cy 5.5TM reporter dyes in combination with a PCR composition containing MUSTANG PURPLETM passive reference dye may be used for a 6-plex multiplex PCR amplification and detection assay.
  • TaqManTM probes with FAMTM, VICTM, ABYTM, JUNTM, CyTM and/or Cy 5.5TM reporter dyes in combination with non-cleavable probes and a PCR composition containing MUSTANG PURPLETM passive reference dye may be used for achieving a multiplex PCR amplification and detection assay greater than a 6-plex.
  • nucleic acid synthesis such as a nucleic acid amplification reaction or a PCR
  • nucleic acid detection e.g., of an amplicon
  • nucleic acid synthesis and nucleic acid detection occur in the same reaction vessel.
  • PCR thermal cycling includes an initial denaturing step at high temperature, followed by a repetitive series of temperature cycles designed to allow template denaturation, primer annealing, and extension of the annealed primers by the polymerase.
  • the samples are heated initially for about 2 to 10 minutes at a temperature of about 95° C to denature the double stranded DNA sample.
  • the samples are denatured for about 10 to 60 seconds, depending on the samples and the type of instrument used.
  • the primers are allowed to anneal to the target DNA at a lower temperature, typically from about 40° C to about 60° C for about 20 to 60 seconds.
  • Extension of the primers by the polymerase is often carried out at a temperature ranging from about 60° C to about 72° C.
  • the amount of time used for extension will depend on the size of the amplicon and the type of enzymes used for amplification and is readily determined by routine experimentation.
  • the annealing step can be combined with the extension step, resulting in a two-step cycling.
  • Thermal cycling may also include additional temperature shifts in PCR assays. The number of cycles used in the assay depends on many factors, including the primers used, the amount of sample DNA present, and the thermal cycling conditions. The number of cycles to be used in any assay may be readily determined by one skilled in the art using routine experimentation.
  • a final extension step may be added after the completion of thermal cycling to ensure synthesis of all amplification products.
  • exemplary thermal cycling conditions for PCR amplifications using the compositions and reaction mixtures disclosed herein are as follows:
  • UNG Step 50 °C, 2 min (e.g., to prevent amplicon/carry over contamination from previous PCRs)
  • the pre-amplification step is truncated prior to reaching an amplification reaction plateau.
  • the methods disclosed herein can include a pre-amplification step which is performed prior to amplification using the compositions and reaction mixtures provided herein.
  • primer may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed.
  • Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature.
  • the nucleic acid may also be obtained from cell culture lines, including transformed and non-transformed cell culture lines.
  • Nucleic acid samples can be extracted from a variety of sources. These include, but are not limited to, for example clothing, soil, paper, metal surfaces, air, water, plant parts, as well as human and/or animal skin, hair, blood, serum, feces, milk, saliva, urine, and/or other secretory or biological fluids.
  • the reverse transcriptase may comprise a mutation as compared to the naturally- occurring reverse transcriptase.
  • the reverse transcriptase may be modified to contain a mutation that provides increased reverse transcriptase stability and/or functionality.
  • Suitable enzymes may also include those in which terminal deoxynucleotidyl transferase (TdT) activity has been reduced, substantially reduced, or eliminated.
  • TdT terminal deoxynucleotidyl transferase
  • an upper bound to an exponential phase of a PCR curve may be established using a derivative method, while a baseline for a PCR curve may be determined to establish a lower bound to an exponential phase of a PCR curve.
  • a threshold value may be established from which a Ct value is determined.
  • Other methods for the determination of a Ct value known in the art for example, but not limited by, various embodiments of a fit point method, and various embodiments of a sigmoidal method (See, e.g., U.S. Patent Nos. 6,303,305: 6,503,720; 6,783,934, 7,228,237 and U.S. Application No. 2004/0096819; the disclosures of which are herein incorporated by reference in their entireties).
  • annealing and “hybridization” are used interchangeably and mean the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure.
  • the primary interaction is base specific, e g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
  • base-stacking and hydrophobic interactions may also contribute to duplex stability.
  • Conditions for hybridizing nucleic acid probes and primers to complementary and substantially complementary target sequences are well known, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, B. Hames and S.
  • annealing takes place is influenced by, among other things, the length of the probes and the complementary target sequences, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization.
  • the preferred annealing conditions depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation.
  • label refers to any atom or molecule which can be used to provide a detectable signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. In some embodiments, the detectable signal is a quantifiable signal.
  • compositions and reaction systems including such compositions for detection of a target nucleic acid in a crude lysate sample by dPCR.
  • the compositions and reaction systems include polymerases, deoxynucleoside triphosphates (dNTP), an aqueous extraction buffer, primers, and/or PCR probes.
  • the compositions and reaction systems are free of a restriction enzyme.
  • the compositions include one or more polymerases.
  • polymerases can be any enzyme capable of replicating a DNA molecule.
  • the compositions may comprise a DNA-dependent DNA polymerase, an enzyme for reverse transcription (RNA-dependent DNA polymerase), and/or a combination of both types of enzymes.
  • RNA-dependent DNA polymerase an enzyme for reverse transcription
  • a combination of DNA dependent DNA polymerases and/or a combination of RNA-dependent DNA polymerase can be present in the compositions disclosed herein.
  • the polymerases as used herein are thermostable DNA polymerases.
  • the thermostable DNA polymerases as used herein are not irreversibly inactivated when subjected to elevated temperatures for the time necessary to effect destabilization of single-stranded nucleic acids or denaturation of double-stranded nucleic acids during nucleic acid synthesis or PCR amplification. Irreversible denaturation of the enzyme refers to substantial loss of enzyme activity.
  • a thermostable DNA polymerase does not irreversibly denature at about 90°-100°C under conditions such as is typically required for PCR amplification.
  • DNA polymerases in accordance with the present teachings can be isolated from natural or recombinant sources, by techniques that are well-known in the art (see, e.g., PCT Publication Nos. WO 92/06200; WO 96/10640; U.S. Patent Nos. 5,455,170; 5,912,155; and 5,466,591, the disclosures of which are fully incorporated herein by reference in their entireties), from a variety of thermophilic bacteria that are available commercially (for example, from American Type Culture Collection, Rockville, Md.) or can be obtained by recombinant DNA techniques (see, e.g., PCT Publication No. WO 96/10640 and U.S. Patent No. 5,912,155).
  • thermostable polymerases Suitable for use as sources of thermostable polymerases or the genes thereof for expression in recombinant systems are, for example, the thermophilic bacteria Thermus thermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosii and other species of the Pyrococcus genus, Bacillus sterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus, and Methanobacterium thermoautotrophicum, and mutants, variants, or derivatives thereof.
  • compositions, systems, methods, and kits provided herein comprise thermostable DNA polymerases selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENTTM DNA polymerase, DEEPVENTTM DNA polymerase, mutants or derivatives thereof having DNA polymerase activity, and any combination of the foregoing.
  • Taq DNA polymerase and mutant forms thereof are commercially available, for example, from Life Technologies (Carlsbad, CA), or can be isolated from their natural source, the (e.g., from the thermophilic bacterium Thermus aquaticus for Taq polymerase), as described previously (see, e.g., U.S. Patent Nos. 4,889,818 and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties).
  • Tne DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga neapolitana (see, e g., PCT Publication No. WO 96/10640 and U.S. Patent No.
  • thermostable polymerases include, but are not limited to, AmpliTaq DNA polymerase and AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific).
  • DNA polymerases from other organisms can also be used herein without departing from the scope or preferred embodiments thereof.
  • DNA polymerases are available commercially from, for example, Life Technologies (Carlsbad, CA), New England BioLabs (Beverly, MA), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, CA), Boehringer Mannheim Biochemicals (Indianapolis, IN) and Perkin Elmer Cetus (Norwalk CT). It is to be understood that a variety of DNA polymerases can be used in the present compositions, methods and kits, including polymerases not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.
  • the compositions provided herein comprise at least one deoxynucleoside triphosphates (dNTP).
  • the composition may further comprise a combination of one or more deoxyribonucleoside triphosphates (dNTPs) and one or more dNTP derivatives.
  • the composition comprises two to eight different dNTPs and/or dNTP derivatives.
  • the composition comprises two, three, four, five, or six different dNTPs and/or dNTP derivatives.
  • dNTPs which can be included in the compositions, reaction mixtures or kits provided herein include, but are not limited to dATP, dCTP, dGTP, dTTP, dUTP, and/or diTP.
  • dNTP derivatives include, but are not limited to, 7-deaza-dGTP (such as 7-deaza-2-deoxy-dGTP), 7-deaza-dATP, alpha-thio-dATP, alpha-thio-dTTP, alpha-thio- dGTP, and/or alpha-thio-dCTP.
  • the composition may further comprise a combination of one or more deoxyribonucleoside triphosphates (dNTPs) and one or more dNTP derivatives.
  • the composition may further comprise one or more dideoxyribonucleoside triphosphates (ddNTPs) and/or one or more ddNTP derivatives.
  • dNTPs, ddNTPs, and derivatives of each thereof are available commercially from sources including Thermo Fisher Scientific, New England Biolabs, and Sigma-Aldrich Company.
  • dNTPs, ddNTPs, and derivatives of each thereof may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g., 3H, 14C, 32P or 35S), vitamins (e g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin (DIG) and the like. Labeled dNTPs, ddNTPs, and derivatives of each thereof may also be obtained commercially, for example from Life Technologies (Carlsbad, CA) or Sigma Chemical Company (Saint Louis, MO).
  • radioisotopes e.g., 3H, 14C, 32P or 35S
  • vitamins e.g., biotin
  • fluorescent moieties e.g., fluorescein, rhodamine, Texas Red, or phy
  • the concentration of individual dNTPs, ddNTP, and/or derivatives of each thereof in the composition need not be identical.
  • dNTPs and/or ddNTPs can be added to give a concentration of each dNTP and/or ddNTP of about .001 mM to about 100 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, or preferably about 0.2 mM to about 0.8 mM, including any concentrations or range of concentrations within any of the forgoing.
  • the compositions comprise one or more primers, which facilitate the synthesis of a DNA molecule (e.g., a single-stranded cDNA molecule or a double-stranded cDNA molecule) complementary to all or a portion of nucleic acid template (RNA or DNA). Additionally, these primers can be used in amplifying nucleic acid molecules in accordance with the present teachings. Oligonucleotide primers can be any oligonucleotide of two or more (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, and so on) nucleotides in length.
  • Exemplary detectable labels include, for instance, a fluorescent dye or fluorphore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like.
  • a fluorescent dye or fluorphore e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence
  • acceptor dyes capable of quenching a fluorescent signal from a fluorescent donor dye
  • Certain PCR inhibitor blocking agents can also be added as a percentage of the final concentration of the composition, for example, from about 0.001% to about 15%, about 0.05% to about 10%, about 0.01% to about 5%, or about 0.1% to about 1%, including any concentrations or range of concentrations within any of the forgoing.
  • the composition can comprise one, two, three, four, five or more different PCR inhibitor blocking proteins and/or agents.
  • the composition comprises an albumin, a fish gelatin and a bovine gelatin.
  • the concentration of each PCR inhibitor blocking protein and/or agent is the same. In some embodiments the concentration of each PCR inhibitor blocking protein and/or agent is different.
  • composition may further comprise crowding agents such as Ficoll 70, glycogen, and polyethylene glycol (PEG).
  • crowding agents such as Ficoll 70, glycogen, and polyethylene glycol (PEG).
  • the buffer concentration of the compositions and/or reaction mixtures as disclosed herein is between about 1 mM and about 500 mM, between about 5 mM and about 250 mM, between about 10 mM and about 200 mM, between about 25 mM and about 150 mM, and between about 50 mM and about 100 mM, including any concentration or range falling within the forgoing amounts. It is to be understood that a wide variety of buffers (or buffer salts) are known in the art that, including those not specifically disclosed herein which can be used in accordance with the present compositions, methods and kits.
  • salts suitable for inclusion in the compositions provided herein include, without limitation, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride and lithium acetate.
  • the composition comprises more than one (e.g., two, three, four, five, six, etc.) salt component.
  • the compositions described herein can comprise two different salts. In other embodiments, the compositions comprise three different salts.
  • the compositions comprise four different salts.
  • the composition comprises a particular salt at a concentration of about 0.5 mM to about 1000 mM, about 1 mM to about 500 mM, about 5 mM to about 250 mM, about 10 mM to about 100 mM, about 5 mM to about 50 mM, and about 2 mM to about 10 mM, including any concentration or range falling within the forgoing ranges.
  • the composition comprises a salt such as potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and/or ammonium acetate
  • the composition comprises the salt at a concentration such that in an assembled PCR is about 5 mM to about 250 mM, 5 mM to about 150 mM, about 10 mM to about 120 mM, about 20 mM to about 100 mM, about 30 mM to about 90 mM, or about 40 to about 80 mM, including any concentration falling within the forgoing ranges.
  • the composition comprises a salt such as magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, or manganese sulfate
  • the composition comprises the salt at a concentration such that in an assembled PCR is about 0.5 mM to about 100 mM, about 1 mM to about 75 mM, about 1 .5 mM to about 50 mM, about 2 mM to about 30 mM, about 3 mM to about 15 mM, about 4 mM to about 10 mM, or about 1 mM to about 5 mM, including any concentration falling within the forgoing ranges.
  • a wide variety of salts or salt solutions are known in the art that can be used in accordance with the present compositions, methods and kits, including those not specifically disclosed herein.
  • the compositions comprise a passive reference control component.
  • the passive reference control minimizes sample-to-sample and/or well-to-well variations in quantitative real-time nucleic acid-detection assays and/or can be included at a concentration allowing its use as detectable control.
  • a reference chromophore specifically a fluorophore, is included as the passive reference control.
  • the reference chromophore is the fluorescent dye.
  • the fluorescent dye is a ROX dye (Thermo Fisher Scientific).
  • the fluorescent dye is a MUSTANG PURPLE dye (Thermo Fisher Scientific).
  • the compositions are reaction mixtures.
  • reaction mixture refers to a mixture of one or more substances and a crude lysate sample which together can cause an amplification reaction; or wherein a mixture of one or more substances and the crude lysate sample can cause a chemical transformation or change.
  • the dNTP of the reaction mixture is selected from dGTP, dCTP, dATP and dTTP.
  • the dNTP derivative of the reaction mixture is selected from 7-deaza-dGTP (such as 7-deaza-2-deoxy-dGTP), 7-deaza-dATP, alpha-thio-dATP, alpha- thio-dTTP, alpha-thio-dGTP, and alpha-thio-dCTP.
  • the reaction mixture comprises a dNTP/dNTP derivative blend comprising some combination of the aforementioned dNTPs and dNTP derivatives.
  • the nucleic acid template of the reaction mixture is DNA.
  • the DNA in the reaction mixture is genomic DNA (gDNA) or complementary DNA (cDNA).
  • the compositions are packaged in a suitable container capable of holding the compositions and which will not significantly interact with components of the compositions.
  • the container can be one designed to permit easy dispensing of the dosage form by individuals or by a liquid handling instrument.
  • the containers of composition can be further packaged into multi-pack units.
  • the multi-pack units may further contain additional containers comprising additives or other reagents to be added to the composition prior to use in a reaction mixture, such as in a PCR.
  • the compositions described herein can be in a liquid form, such as in a hydrated solution. In other embodiments, the compositions described herein can be in a gel form.
  • a “gel” as used herein is a composition which is not solid or frozen at -20°C. In yet other embodiments, the compositions described herein can be in a dehydrated or dried form, such as in a lyophilized composition.
  • the kit further comprises, in addition to the composition or master mix, at least one primer pair specific for PCR amplification of a nucleic acid target, and/or at least one probe specific for the amplified nucleic acid target.
  • the probe can be a TaqManTM probe, a HydrolEasyTM probe, a minor groove binding (MGB) probe, a locked nucleic acid (LNA) probe, a SYBR Green or SYBR GreenERTM probe, or a cycling probe technology (CPT) probe.
  • the extraction buffer is an aqueous extraction buffer.
  • the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.
  • the crude lysate sample is an unpurified lysate sample.
  • the preparing of the crude lysate sample and the performing of the amplification reaction are performed without extracting the crude biological sample or the crude lysate sample with a chloroform-based compound and/or a phenol-based compound.
  • the method is performed without incubating the crude biological sample or the crude lysate sample with a restriction enzyme.
  • the extraction buffer is an aqueous extraction buffer.
  • the preparing of the crude lysate sample further comprises centrifuging the mixture of the homogenized crude biological sample and the extraction buffer to generate a supernatant; and collecting the supernatant to generate the crude lysate sample.
  • the reaction mixture is an unpurified reaction mixture.
  • the crude lysate sample is an unpurified biological sample.
  • the preparing of the crude lysate sample and the performing of the amplification reaction are performed without extracting the crude biological sample or the crude lysate sample with a chloroform-based compound and/or a phenol-based compound.
  • the loading of the reaction mixture includes transferring a portion of the reaction mixture including the crude lysate sample into a plurality of microchambers of the microfluidic array plate, and the performing of the amplification reaction includes thermocycling the reaction mixture in the plurality of microchambers.
  • the microfluidic array plate is a single plate.
  • the crude lysate sample is an environmental sample in the form of at least one selected from a water sample, an air sample, a plant sample, a fungal sample, and a soil sample.
  • the reaction mixture further includes a reverse transcriptase.
  • the target nucleic acid belongs to a pathogen infecting a host or subject.
  • the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.
  • the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.
  • the crude biological sample is prepared by homogenizing or grinding a crude biological sample including the biological tissue, and mixing the homogenized biological sample with an extraction buffer.
  • the extraction buffer is a lysis buffer.
  • the amplification reaction is performed without extracting the crude lysate sample with a chloroform -based compound and/or a phenol-based compound.
  • the amplification reaction is performed on a microfluidic array plate of a digital PCR instrument.
  • the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.
  • the crude lysate sample is an environmental sample in the form of at least one selected from a water sample, an air sample, a plant sample, a fungal sample, and a soil sample.
  • a digital polymerase chain reaction (PCR) kit comprises a first mixture including an extraction buffer for producing a crude lysate sample, the crude lysate sample having biological tissue; and a second mixture including an amplification mixture for amplification of a target nucleic acid in the crude lysate sample, wherein the amplification mixture includes at least one probe configured to hybridize to amplicons of the target nucleic acid, and at least one primer set configured to synthesis the amplicons, at least one deoxynucleoside triphosphate (dNTP), and at least one polymerase configured to incorporate the at least one deoxynucleoside triphosphate (dNTP) for synthesizing the amplicons.
  • dNTP deoxynucleoside triphosphate
  • the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.
  • the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.
  • the crude lysate sample is prepared by homogenizing or grinding a crude biological sample including the biological tissue, and mixing the homogenized crude biological sample with the extraction buffer.
  • the extraction buffer is a lysis buffer.
  • the extraction buffer is an aqueous extraction buffer.
  • the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.
  • the crude lysate sample is an unpurified biological sample.
  • the crude lysate sample is a biological tissue sample in the form of at least one selected from plasma, serum, biological fluids, semen, saliva, whole blood, feces, milk, organ and hair.

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Abstract

Procédé de réalisation d'une réaction en chaîne de la polymérase numérique (dPCR) comprenant la réalisation d'une réaction d'amplification sur un mélange réactionnel comprenant un échantillon de lysat brut pour générer des amplicons d'un acide nucléique cible d'un agent pathogène, l'échantillon de lysat brut comprenant un tissu biologique. La fluorescence est détectée à partir d'une sonde hybridée aux amplicons. Un procédé de détection d'un agent pathogène chez un sujet comprend la mise en oeuvre de la réaction d'amplification susmentionnée. L'échantillon de lysat brut comprend le tissu biologique prélevé sur le sujet. La fluorescence détectée à partir de la sonde hybridée aux amplicons est utilisée pour déterminer si l'agent pathogène est présent chez le sujet. L'invention concerne également des compositions, des systèmes de PCR et des kits.
PCT/US2025/011220 2024-01-11 2025-01-10 Extraction d'échantillon de lysat brut pour pcr numérique Pending WO2025151807A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2025257040A1 (fr) * 2024-06-12 2025-12-18 Qiagen Gmbh Procédé de détection d'un pathogène viral dans un produit laitier ou de l'eau

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