Crude lysate sample extraction for digital PCR

Abstract

A method of performing a digital polymerase chain reaction (dPCR) includes 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 lysate sample includes biological tissue. Fluorescence is detected from a probe hybridized to the amplicons. A method of detecting pathogen in a subject includes performing the above amplification reaction. The crude lysate sample includes biological tissue from the subject. The fluorescence detected from the probe hybridized to the amplicons is used to determine if the pathogen is present in the subject. Compositions, PCR systems, and kits are also provided.

Description

CRUDE LYSATE SAMPLE EXTRACTION FOR DIGITAL PCRCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 620,126, titled “Crude Lysate Sample Extraction for digital PCR,” filed January 11, 2024, the entirety of which is incorporated herein by this reference.FIELD

[0002] 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).BACKGROUND

[0003] For many medical, diagnostic, and forensic applications, 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.

[0004] For example, there is a need for sensitive detection of plant pathogens. 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). X. 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. Thus, 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.

[0006] Polymerase chain reaction (PCR) is an in vitro method for the enzymatic synthesis of specific nucleic acid sequences in target nucleic acid(s). A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by a polymerase results in the exponential accumulation of a specific fragment. PCR can selectively amplify a specific nucleic acid sequence by several orders of magnitude.

[0007] Molecular detection of plant pathogens is conventionally performed by quantitative PCR (qPCR). However, qPCR can require lengthy purification steps to eliminate PCR inhibitors present in the plant tissue and therefore is not compatible with crude lysate samples.

[0008] FIG. 5 shows a conventional sample preparation and amplification workflow requiring a purification step.

[0009] Referring to FIG. 5, 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.

[0010] Then, in 509, chloroform (such as phenol: chloroform: isoamyl alcohol) is added to the mixture to extract nucleic acid from the crude sample, and centrifuged. An aqueous phase is retained, and the organic phase is discarded.

[0011] Next, in 51 1, 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.

[0012] 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.

[0013] The ethanol is removed, and the pellet of purified nucleic acid is dissolved in a desired buffer or solution.

[0014] Then, 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.

[0015] FIG. 6 is another conventional sample preparation and amplification workflow requiring a purification step.

[0016] Referring to FIG. 6, 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.

[0017] After vortexing, the mixture is incubated for an amount of time sufficient for lysing in 607.

[0018] Next, in 611, 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.

[0019] 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.

[0020] The ethanol is removed, and the pellet of purified nucleic acid is dissolved in a desired buffer or solution.

[0021] Then, 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 purifiedsample 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.

[0022] 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. In the case of detection of a pathogen in a host, incubating the sample from the host with a restriction enzyme prior to amplification can prevent replication of non-targeted nucleic acid, thus leading to better detection of the target nucleic acid(s). However, the restriction enzymes can be costly.

[0023] FIGS. 7 shows a conventional sample preparation and amplification workflow using a restriction enzyme.

[0024] Referring to FIG. 7, 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.

[0025] Then, 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.

[0026] Conventionally, qPCR is not reliable for detection at the early stages of the infection (for instance, prior to about 8 weeks from initial exposure to A fastidiosd). Furthermore, the kits and enzymes for such nucleic acid purification and extraction methods are expensive and / or lengthy.SUMMARY

[0027] Methods, compositions, polymerase chain reaction (PCR) systems, and kits are provided for the extraction, amplification, detection, and / or quantitation of nucleic acids from abiological sample by digital polymerase chain reaction (dPCR). In embodiments, the biological sample is a crude lysate sample.

[0028] 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.

[0029] In the first embodiment, 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.

[0030] In the first embodiment, the method is performed without incubating the crude lysate sample with a restriction enzyme.

[0031] In the first embodiment, the extraction buffer is an aqueous extraction buffer.

[0032] In the first embodiment, the extraction buffer is a lysis buffer.

[0033] In the first embodiment, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0034] In the first embodiment, 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.

[0035] In the first embodiment, the reaction mixture is an unpurified reaction mixture.

[0036] In the first embodiment, the crude lysate sample is an unpurified lysate sample.

[0037] In the first embodiment, 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.

[0038] In the first embodiment, 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.

[0039] In the first embodiment, 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.

[0040] In the first embodiment, the microfluidic array plate is a single plate.

[0041] In the first embodiment, the method further comprises quantifying an amount of the target nucleic acid based on the fluorescence detected.

[0042] In the first embodiment, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0043] In the first embodiment, 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.

[0044] In the first embodiment, 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.

[0045] In the first embodiment, 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.

[0046] In the first embodiment, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0047] In a second embodiment, a method of detecting pathogen in a subject, comprises performing an amplification reaction on a reaction mixture including a crude lysate sample to generate amplicons of a target nucleic acid of the pathogen, wherein the crude lysate sample includes biological tissue from the subject; and detecting fluorescence from a probe hybridized to the amplicons to determine if the pathogen is present in the subject.

[0048] In the second embodiment, the presence of the pathogen is determined before the subject is symptomatic of an infection by the pathogen.

[0049] In the second embodiment, 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.

[0050] In the second embodiment, the method is performed without incubating the crude biological sample or the crude lysate sample with a restriction enzyme.

[0051] In the second embodiment, the extraction buffer is an aqueous extraction buffer.

[0052] In the second embodiment, the extraction buffer is a lysis buffer.

[0053] In the second embodiment, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0054] In the second embodiment, 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.

[0055] In the second embodiment, the reaction mixture is an unpurified reaction mixture.

[0056] In the second embodiment, the crude lysate sample is an unpurified biological sample.

[0057] In the second embodiment, 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.

[0058] In the second embodiment, 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.

[0059] In the second embodiment, 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.

[0060] In the second embodiment, the microfluidic array plate is a single plate.

[0061] In the second embodiment, the method further comprises quantifying an amount of the target nucleic acid based on the fluorescence detected.

[0062] In the second embodiment, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0063] In the second embodiment, 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.

[0064] In the second embodiment, 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.

[0065] In the second embodiment, 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.

[0066] In the second embodiment, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0067] In a third embodiment, a polymerase chain reaction (PCR) system, comprises a reaction mixture including a crude lysate sample, at least one probe configured to hybridize to amplicons of a 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 add the at least one deoxynucleoside triphosphate (dNTP) for synthesizing the amplicons, wherein the PCR system is used to perform an amplification reaction on the reaction mixture including the crude lysate sample to generate the amplicons of the target nucleic acid, and wherein the crude lysate sample includes biological tissue.

[0068] In the third embodiment, 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.

[0069] In the third embodiment, the reaction mixture further includes a reverse transcriptase.

[0070] In the third embodiment, the target nucleic acid belongs to a pathogen infecting a host or subject.

[0071] In the third embodiment, presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

[0072] In the third embodiment, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0073] In the third embodiment, the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

[0074] In the third embodiment, 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.

[0075] In the third embodiment, the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

[0076] In the third embodiment, the extraction buffer is a lysis buffer.

[0077] In the third embodiment, the extraction buffer is an aqueous extraction buffer.

[0078] In the third embodiment, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0079] In the third embodiment, the reaction mixture is an unpurified reaction mixture, and / or the crude lysate sample is an unpurified biological sample.

[0080] In the third embodiment, 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.

[0081] In the third embodiment, the amplification reaction is performed without extracting the crude lysate sample with a chloroform-based compound and / or a phenol -based compound.

[0082] In the third embodiment, the amplification reaction is performed on a microfluidic array plate of a digital PCR instrument.

[0083] In the third embodiment, the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

[0084] In the third embodiment, the microfluidic array plate is a single plate.

[0085] In the third embodiment, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0086] In the third embodiment, 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.

[0087] In the third embodiment, 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.

[0088] In the third embodiment, 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.

[0089] In a fourth embodiment, 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.

[0090] In the embodiment aspect, 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.

[0091] In the fourth embodiment, the reaction mixture further includes a reverse transcriptase.

[0092] In the fourth embodiment, the target nucleic acid belongs to a pathogen infecting a host or subject.

[0093] In the fourth embodiment, presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

[0094] In the fourth embodiment, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0095] In the fourth embodiment, the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

[0096] In the fourth embodiment, 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.

[0097] In the fourth embodiment, the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

[0098] In the fourth embodiment, the extraction buffer is a lysis buffer.

[0099] In the fourth embodiment, the extraction buffer is an aqueous extraction buffer.

[0100] In the fourth embodiment, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0101] In the fourth embodiment, wherein the second mixture is mixed with the crude biological sample to produce a reaction mixture.

[0102] In the fourth embodiment, the reaction mixture is an unpurified reaction mixture.

[0103] In the fourth embodiment, the crude lysate sample is an unpurified biological sample.

[0104] In the fourth embodiment, 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.

[0105] In the fourth embodiment, 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.

[0106] In the fourth embodiment, the target nucleic acid is amplified on a microfluidic array plate of a digital PCR instrument.

[0107] In the fourth embodiment, the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

[0108] In the fourth embodiment, the microfluidic array plate is a single plate.

[0109] In the fourth embodiment, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0110] In the fourth embodiment, 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.

[0111] In the fourth embodiment, 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.

[0112] In the fourth embodiment, 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.

[0113] The above-described embodiments and other features are exemplified by the following figures and detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Embodiments will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. FIGS. 1-8 represent non-limiting, example embodiments as described herein.

[0115] 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”).

[0116] 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”).

[0117] 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 incubationstep prior to dPCR (“Omins”) and as a purified sample with a conventional restriction enzyme incubation step prior to dPCR (“30mins 35°C”).

[0118] 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”).

[0119] FIG. 2A shows copy number detection levels in the dPCR assay of FIG. 1 A for the purified sample.

[0120] FIG. 2B shows copy number detection levels in the dPCR assay of FIG. 1 A for the crude lysate sample.

[0121] FIG. 2C shows copy number detection levels in the dPCR assay of FIG. IB for the purified sample.

[0122] FIG. 2D shows copy number detection levels in the dPCR assay of FIG. IB for the crude lysate sample.

[0123] FIG.3A shows copy number detection levels in the dPCR assay of FIG. 1C for the purified sample.

[0124] FIG. 3B shows copy number detection levels in the dPCR assay of FIG. 1C for the crude lysate sample.

[0125] FIG. 3C shows copy number detection levels in the dPCR assay of FIG. ID for the purified sample.

[0126] FIG. 3D shows copy number detection levels in the dPCR assay of FIG. ID for the crude lysate sample.

[0127] 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.

[0128] FIGS. 5, 6 and 7 show conventional sample preparation and amplification workflows.

[0129] FIG. 8 shows a simplified, streamlined sample preparation and amplification workflow according to embodiments.DETAILED DESCRIPTION

[0130] Various embodiments will now be described more fully with reference to the accompanying drawings in which some embodiments are shown. However, specific functional details disclosed herein are merely representative for purposes of describing embodiments. Thus, the invention may be embodied in many alternate forms and should not be construed as limited to only embodiments set forth herein. Therefore, it should be understood that there is no intent to limit embodiments to the particular forms disclosed, but on the contrary, embodiments are to cover all modifications, equivalents, and alternatives falling within the scope.

[0131] In the drawings, like numbers refer to like elements throughout the description of the figures.

[0132] Although the terms 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. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0133] It will be understood that, if an step is referred to as being “directly after” another step, there are no intervening steps present. Other words used to describe the relationship between steps should be interpreted in a like fashion (e.g., “between” versus “directly between,” “before” versus “directly before,” etc.).

[0134] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes” and / or “including,” if used herein, specify the presence of stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0135] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.[00136J In order to more specifically describe embodiments, various features will be described in detail with reference to the attached drawings. However, embodiments described are not limited thereto.

[0137] Provided herein are methods, compositions, polymerase chain reaction (PCR) systems, and kits for the extraction, amplification, detection, and / or quantitation of nucleic acids from a biological sample by digital polymerase chain reaction (dPCR). In some embodiments, the biological sample is a crude lysate.

[0138] Digital polymerase chain reaction (dPCR) is a specific type of PCR and used to detect and quantify target nucleic acids.

[0139] Both dPCR and real-time or quantitative PCR (qPCR) can be used to detect and quantify nucleic acids. While these technologies share similarities, key differences in quantification methods confer each tool with application-dependent benefits or limitations. For example, qPCR provides a broad dynamic range, but dPCR provides greater precision.

[0140] 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.

[0141] 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 arun at a limiting dilution to ensure at least one reaction does not contain any target nucleic acid(s). Advantages of dPCR include 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.

[0142] In dPCR according to embodiments, 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.[00143J The following factors may influence the selection of a dPCR system over a quantitative polymerase chain reaction (qPCR) system for detection of microbial targets: (i) dPCR provides absolute quantification, (ii) dPCR has a high inhibitor tolerance (i.e., detection of pathogens such as Xylella fastidiosa (bacteria) DNA from insect vectors), (iii) dPCR has a low limit of detection of microbes in samples such as plant, human, animal, environmental samples, and (iv) dPCR permits analysis of rare or limited field samples. Furthermore, in addition to a combination of the buffers and reagents, the small reaction size required for dPCR systems (such as the Absolute Q™ Digital PCR System by Thermo Fisher Scientific) helps mitigate any inhibition when performing qPCR that utilizes larger sample volumes.

[0144] In exemplary embodiments, a method surprisingly detects infections at an earlier stage than conventional PCR methods. In some embodiments, 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. However, embodiments are not limited thereto. Thus,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.

[0145] In some embodiments, 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.

[0146] In dPCR applications using a conventional restriction enzyme incubation step, 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.

[0147] FIG. 8 shows a simplified, streamlined sample preparation and amplification workflow according to embodiments.

[0148] Referring to FIG. 8, a crude lysate sample is prepared by homogenizing or grinding the crude sample, and mixing the crude sample with an extraction buffer in 810.

[0149] Then, 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.

[0150] In embodiments, dPCR is performed on the crude lysate sample directly after preparing the crude lysate sample. That is, the crude lysate sample is not subjected to any further treatments (such as additional lysing, extraction, incubation, etc.) prior to performing dPCR.

[0151] In some embodiments, the method is a dPCR workflow process for preparing and utilizing a crude lysate sample. In some embodiments, 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. In other embodiments, 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.

[0152] In some embodiments, the microfluidic array plate is a QuantStudio™ Absolute Q™ MAP 16 plate (Thermo Fisher Scientific).

[0153] In some embodiments, the preparing of the crude lysate sample includes homogenizing or grinding sample tissue in an aqueous extraction buffer. In some embodiments, 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.

[0154] In some embodiments, 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.

[0155] 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.

[0156] In some embodiments, 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.

[0157] In some embodiments, 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).

[0158] In some embodiments, 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. These advances highlight the potential of the streamlined method in addressing disease detection and monitoring challenges in diagnostic research and surveillance efforts.

[0159] In some embodiments, the methods disclosed herein are useful in a wide range of applications and assays that involve detecting, quantitating, and / or characterizing target nucleic acids.

[0160] In exemplary embodiments, the methods, compositions, reaction systems, and kits are used on a crude sample. As used herein, the term “crude sample” refers to a biological sample that has not been subjected to an organic extraction or purification to provide a purified nucleicacid sample. Tn some embodiments, the biological sample is subjected to homogenizing / grinding and extraction with an aqueous extraction buffer to form the crude lysate sample. In some embodiments, 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.

[0161] In some embodiments, 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. Each product is primed using a distinct primer pair. A multiplex reaction may further include labeled probes specific to each product, wherein the probes are detectably labeled with different detectable moieties. In some embodiments, 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.

[0162] In some embodiments, the amplification or reaction mixture includes “hot start” components or steps to further prevent, reduce or eliminate nonspecific nucleic acid synthesis. Several methods exist for performing hot start reactions including manual techniques, barriers, reversible polymerase inactivation, and specially-designed hairpin primers. In some embodiments, 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. Such examples of hot start components can include, but are not limited to, for example, an antibody, a chemical modification (e.g., of the polymerase), an oligonucleotide, an aptamer, a specially- designed primer, a binding protein, and / or a sequestration wax bead. Wax beads for hot start PCR are commercially available, e.g., HotStart ™ Storage reaction tubes (Thermo Fisher Scientific). Selection of a suitable hot start aptamer can be performed by a method known in the art or a commercially available hot start aptamer can be used. Similarly, selection of a suitablehot start hairpin primer can be performed by a method known in the art or a commercially available hot start primer can be used. Antibodies for hot start PCR can be generated or selected by various methods known in the art. Alternatively, 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. In some embodiments, 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. Some of the hot start mechanisms, components or steps are described in more detail below.

[0163] In general, manual hot start methods usually, though not always, require the researcher to withhold a critical component, usually magnesium or the polymerase, until the reaction has been heated. The withheld component then is added to initiate the reaction. A second method uses a physical barrier (e.g., wax) to separate a critical component from the template and primers. U.S. Pat. No. 5,565,339 describes using a wax barrier to separate the various PCR reagents from each other in a test tube. U.S. Pat. No. 5,413,924 describes using a paraffin wax bead to sequester the DNA polymerase.

[0164] 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. For example, 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. In another example, 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.

[0165] Another approach to achieve hot start amplification is to design primers that will selfanneal to form specific hairpin structures. The hairpin primers will not be able to anneal to the target nucleic acid while in the hairpin conformation. The hairpin primers will remain in a hairpin conformation until heated to a denaturation temperature. However, if the hairpin structureincludes a single-strand extension, then the hairpin structure itself resembles a primer annealed to a template and can result in strand extension.

[0166] Various other 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. In certain embodiments, 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.

[0167] In some embodiments, 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. During the extension or elongation phase of a PCR reaction, 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.

[0168] The terms "upstream" and "downstream" are used herein in relation to the synthesis of the nascent strand that is primed by a target-specific primer. Thus, for example, 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.

[0169] 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. In the TaqMan® 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. When this occurs, the reporter fluorophore is released from the probe, and subsequently, is no longer in close proximity to the quencher. This produces a perpetual increase in reporter signal with each extension phase as the PCR reaction continues cycling. In order to achieve maximal signal with each cycle, 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. In some embodiments, the methods disclosed herein involve the use of a TaqMan assay for nucleic acid analysis and / or detection. For example, the compositions of the present disclosure can be used in methods involving TaqMan probes and / or assays. Such assays can include, but are not limited to gene expression assays (e.g., TaqMan™ Gene Expression Assays), copy number variation assays (e g., TaqMan™ Copy Number Assays), genotyping assays (e.g., TaqMan™ Drug Metabolism Genotyping Assays or TaqMan™ SNP Genotyping Assays), miRNA assays (e.g., TaqMan™ MicroRNA Assays) or RNA quantitation assays (e.g., two-step reverse transcription-polymerase chain reaction assays), and TaqMan™ Low Density Array Assays.

[0170] As used herein, the term “reaction vessel” generally refers to any container in which an amplification reaction can occur in accordance with the present teachings. In some embodiments, a reaction vessel may be a microtube, for example, but not limited to, a 0.2 mL or a 0.5 mL reaction tube such as a MicroAmp™ Optical tube (Applied Biosystems™, Thermo Fisher Scientific) or a micro-centrifuge tube, or other containers of the sort in common practice in molecular biology laboratories. In some embodiments, 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 QuantStudio™ Absolute Q™ MAP 16 plate (Thermo Fisher Scientific), a TaqMan™ Array plate (Applied Biosystems™; Thermo Fisher Scientific), a spot on a glass slide, a well in an Applied Biosystems™ TaqMan™ Array Card or Plate (Thermo Fisher Scientific) or a through-hole of an Applied Biosystems™ TaqMan™ OpenArray™ plate (Thermo Fisher Scientific). For example,a plurality of reaction vessels may reside on the same support. In some embodiments, lab-on-a- chip-like devices, available for example from Caliper and Fluidigm, can provide for reaction vessels. In some embodiments, 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.

[0171] The terms “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. In certain embodiments, the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.

[0172] As used herein, the term “amplifying” refers to any means by which at least a part of a target polynucleotide, target polynucleotide surrogate, or combinations thereof, is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Any of several methods can be used to amplify the target polynucleotide. Any in vitro means for multiplying the copies of a target sequence of nucleic acid can be utilized. These include linear, logarithmic, or any other amplification method. 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. 19:225-232 (1998); and Baner et al. Nucleic Acid Res. 26: 5073-5078 (1998)), and strand displacement amplification (SDA) (Little et al. Clin. Chem. 45:777-784 (1999)), among others. Many systems are suitable for use in amplifying target nucleic acids and are contemplated herein as would be understood by one of skill in the art. In some embodiments of the present methods, nucleic acid amplification reaction can be performed by PCR. In some embodiments, the PCR can be endpoint PCR. In some embodiments, the PCR can be digital PCR. In yet other embodiments, the PCR can comprise thermal cycling. In some embodiments, the thermal cycling can be optimized for fast thermal cycling.

[0173] In some embodiments, 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. In some embodiments, 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.

[0174] In some embodiments, 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. In some embodiments, one of the multiplexed targets is an endogenous or an exogenous internal positive control for amplification. In some embodiments, the methods for detecting a target nucleic acid can be multiplex PCR and / or detection assays in which multiple targets are simultaneously detected. In certain embodiments, 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. In an embodiment, one of the multiplexed targets is an endogenous or an exogenous internal positive control for amplification and / or detection. In some embodiments, a multiplicity of targets are simultaneously amplified and detected. In some embodiments, a multiplicity of targets are amplified and detected in the same reaction vessel. In some embodiments, various TaqMan™ probe reporter dye and passive dye options can be combined for multiplex PCR. For example, TaqMan™ probes with FAM™, VIC™, and ABY™ 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. For another example, TaqMan™ probes with FAM™, VIC™, ABY™, and JUN™ reporter dyes in combination with a PCR composition containing MUSTANG PURPLE™ passive reference dye may be used for a 4-plex multiplex PCR amplification and detection assay. For another example, TaqMan™ probes with FAM™, VIC™, ABY™, JUN™, Cy™ and Cy 5.5™ reporter dyes in combination with a PCR composition containing MUSTANG PURPLE™ passive reference dye may be used for a 6-plex multiplex PCR amplification and detection assay. For another example, TaqMan™ probes with FAM™, VIC™, ABY™, JUN™, Cy™ and / or Cy 5.5™ reporter dyes in combination with non-cleavableprobes and a PCR composition containing MUSTANG PURPLE™ passive reference dye may be used for achieving a multiplex PCR amplification and detection assay greater than a 6-plex. In some embodiments, nucleic acid synthesis (such as a nucleic acid amplification reaction or a PCR) and nucleic acid detection (e.g., of an amplicon) can occur simultaneously. Accordingly, in some embodiments, nucleic acid synthesis and nucleic acid detection occur in the same reaction vessel.[00175J In general, 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. Generally, 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. Then, in the beginning of each cycle, the samples are denatured for about 10 to 60 seconds, depending on the samples and the type of instrument used. After denaturing, 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.Additionally, 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. Optionally, a final extension step may be added after the completion of thermal cycling to ensure synthesis of all amplification products.

[0176] In one embodiment, exemplary thermal cycling conditions for PCR amplifications using the compositions and reaction mixtures disclosed herein are as follows:UNG Step (Optional): 50 °C, 2 min (e.g., to prevent amplicon / carry over contamination from previous PCRs)Activation: 95 °C, 20 secDenaturation: 95 - 97 °C / l-3 secExtension: 60 - 62 °C / 20 - 30 sec (x 40 cycles).

[0177] The composition and reaction mixtures provided may also be used for amplification reactions in which a limited number of cycles occur (for example, but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cycles of amplification), such as in pre-amplification reactions. In certain embodiments, a pre-amplification step is performed using the provided compositions and reaction mixtures (see, for e.g., US Patent No. 9, 206, 475, the disclosure of which is incorporated by reference in its entirety). In some embodiments, a pre-amplification is performed with a pair of universal forward and reverse primers. In some embodiments, preamplification is performed for under than 20 cycles, for example, 2-18 cycles. In some embodiments, the pre-amplification step is truncated prior to reaching an amplification reaction plateau. In some embodiments, the methods disclosed herein can include a pre-amplification step which is performed prior to amplification using the compositions and reaction mixtures provided herein.

[0178] PCR with the disclosed composition can be performed on “standard” digital PCR instrumentation, e.g., a QuantStudio™ Absolute Q™ Digital PCR System (Thermo Fisher Scientific), a Digital Droplet PCR System (BioRad), Digital LightCycler™ dPCR System (Roche), or a QIAcuity™ Digital PCR System (Qiagen), Naica™ System (Stilla) . However, embodiments are not limited thereto.

[0179] In some embodiments, the systems, kits, and methods provided herein offer an advantage over standard or traditional systems, kits and methods (such as other commercially available master mixes or kits) used for nucleic acid synthesis and / or detection. In some embodiments, such advantages include, but are not limited, to any of the following: a) can be used to amplify nucleic acids (e.g., by dPCR) directly from minimally- processed lysate samples (e.g., can amplify targets directly from lysates of saliva and whole blood) b) improves the accuracy of copy number variation results; c) provides increased tolerance to various PCR inhibitors; e) provides increased specificity and sensitivity; f) can be used with fast thermal cycling protocols for quicker read-outs; and / org) allows for the capability to multiplex (e.g., (i) can amplify a multiplicity of targets using a single sample; two or more targets in one sample or (ii) can amplify multiple samples; two targets in two different samples) in a single reaction at substantially the same time.

[0180] In some embodiments, the accuracy of copy number variation is improved by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% or at least 25% when using the compositions, kits, and methods provided herein according to embodiments compared to copy number variation observed when performing qPCR using standard or traditional compositions, kits, and method. In some embodiments, the input detection sensitivity is increased by at least 10 fold, 50 fold, 100 fold, 500 fold, or 1000 fold when using the compositions, kits, and methods provided herein according to embodiments compared to the input detection sensitivity observed when performing qPCR using standard or traditional compositions, kits, and method. In some embodiments, the specificity and sensitivity is at least 80%, 85%, 90%, 95% or 99% accurate for genotyping when using the compositions, kits, and methods provided herein according to embodiments. In some embodiments, any of the advantages (a) through (g), listed above, are at least comparable to those observed using standard or traditional compositions, kits and methods for nucleic acid synthesis and / or detection.

[0181] PCR “master mixes” improve the efficiency of amplification reactions. These master mixes contain a combination of reagents common to most PCR reactions, such as a buffer, a salt such as MgCh, deoxynucleotide triphosphates (dNTPs), and a polymerase (such as a DNA polymerase or a RNA polymerase). When performing PCR, each reaction volume includes the master mix and a specific target nucleic acid and primer pair. Typically, master mixes are manufactured and distributed as concentrated solutions or lyophilized powders which are subsequently diluted or dissolved when final reactions are assembled.

[0182] As used herein, “nucleotide” refers to a base-sugar-phosphate combination. A “nucleoside” refers to a base-sugar combination. Nucleotides are monomeric units of a nucleic acid sequence (e.g., DNA and RNA). The term nucleotide includes mono-, di- and triphosphate forms of deoxyribonucleosides and ribonucleosides and their derivatives. dNTPs may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g., H-3, C-14, P-32 or S-35), vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin andthe like. Labeled dNTPs may be obtained commercially, for example from Thermo Fisher Scientific or Sigma-Aldrich Company.

[0183] As used herein, “polynucleotide” and “oligonucleotide” refer to a synthetic or biologically produced molecule comprising a covalently linked sequence of nucleotides which may be joined by a phosphodiester bond between the 3' position of the pentose of one nucleotide and the 5' position of the pentose of the adjacent nucleotide. In addition, a polynucleotide or oligonucleotide may contain modified or non-naturally occurring sugar residues (e.g., arabinose) and / or modified base residues. A polynucleotide or oligonucleotide may also comprise blocking groups that prevent the interaction of the molecule with particular proteins, enzymes or substrates.

[0184] As used herein, “nucleic acid” includes compounds having a plurality of natural nucleotides and / or non-natural (or “derivative”) nucleotide units. A “nucleic acid” can further comprise non-nucleotide units, for example peptides. “Nucleic acid” therefore encompasses compounds such as DNA, RNA, peptide nucleic acids, phosphothioate-containing nucleic acids, phosphonate-containing nucleic acids and the like. There is no particular limit as to the number of units in a nucleic acid, provided that the nucleic acid contains 2 more nucleotides, nucleotide derivatives, or combinations thereof, specifically 5, 10, 15, 25, 50, 100, or more. Nucleic acids can encompass both single and double-stranded forms, and fully or partially duplex hybrids (e.g., RNA-DNA, RNA-PNA, or DNA-PNA).

[0185] As used herein, the term “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 primer is preferably single-stranded for maximum efficiency in amplification. A primer is typically 11 bases or longer; more specifically, a primer is 17 bases or longer, although shorter or longer primers may be used depending on the need. As will be appreciatedby those skilled in the art, the oligonucleotides may be used as one or more primers in various extension, synthesis or amplification reactions.

[0186] The complement of a nucleic acid sequence as used herein refers to an oligonucleotide or a polynucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength, and incidence of mismatched base pairs.

[0187] Stability of a nucleic acid duplex is measured by the melting temperature (“Tm”). The Tm of a particular nucleic acid duplex under specified conditions is the temperature at which half of the base pairs have disassociated.

[0188] When referring to a thermostable DNA polymerase, one unit of activity is the amount of enzyme that will incorporate 10 nanomoles of dNTPs into acid-insoluble material (i.e., DNA or RNA) in 30 minutes under standard primed DNA synthesis conditions.

[0189] As used herein, the term “target,” “target sequence,” “target template,” “target nucleic acid” or “target nucleic acid sequence” refers to a nucleic acid sequence or region of a nucleic acid which is to be either amplified, detected, or both. Typically, in a PCR, the target sequence resides between the two primer sequences used for amplification.

[0190] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0191] Exemplary samples of nucleic acids include DNA and / or RNA. In one embodiment, the nucleic acid is not isolated or purified. In some embodiments, the nucleic acid sample is within a biological sample. For example, the nucleic acid may be present in a complex mixture, such as a crude lysate or whole cell extract. In another embodiment, the nucleic acid may be in situ and exist within its normal cellular, bacterial, or viral environment.

[0192] The target nucleic acid may be obtained from any source and may comprise any number of different compositional components. For example, the target may be a nucleic acid (e.g., DNA or RNA), messenger RNA (mRNA), transfer RNA (tRNA), small interfering RNA (siRNA), microRNA (miRNA), or other mature small RNA, and may comprise nucleic acid analogs or other nucleic acid mimics. The target may be methylated, non-methylated, or both. Further, it will be appreciated that “target nucleic acid” may refer to the target nucleic acid itself, as well as surrogates thereof, for example, amplification products and native sequences.

[0193] The nucleic acid samples of the present teachings may be obtained from any number of biological sources, including without limitation, viruses, archae, protists, prokaryotes and eukaryotes, for example, from a biological sample obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. The nucleic acid may be obtained from cells, tissues, organs, or organisms in different developmental stages. The nucleic acid samples may also be obtained from cancer cells and precancerous cells obtained from animals, including humans. 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.

[0194] “Biological sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, platelets, red blood cells, and the like), lymph, bone marrow, sputum, bronchoalveolar lavage, amniotic fluid, hair, skin, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. Prior to target nucleic acid preparation,biological samples may be fresh, frozen or formalin- or paraformalin-fixed paraffin-embedded tissue (FFPE).

[0195] The PCR compositions can tolerate PCR inhibitors that are carried over from sample preparation. The PCR compositions are compatible with crude cell or tissue lysates such as those prepared from, for example, whole blood in liquid form, whole blood dried on paper, buccal swabs (e.g., oral mucosa), raw saliva, urine, serum, hair follicles, plant leaf, and FFPE.

[0196] As used herein, the terms “crude sample” or “crude biological sample” refers to a specimen of biological origin suspected of containing nucleic acids, which has not undergone procedures for the isolation or purification of those nucleic acids. For example, a sample of blood or urine is a crude sample. A buccal swab of cheek cells is another example of a crude sample. Crude samples include, but are not limited to, blood, diluted blood, blood on paper, buccal swabs, and buccal swabs on a substrate for samples storage, such as FTA paper. The cells in a crude sample are lysed to create a “crude lysate sample.” One of skill in the art will recognize an enormous variety of other crude samples or crude lysates whose analysis would be facilitated by the present teachings.

[0197] Following amplification or synthesis, the amplified nucleic acid fragments may be isolated for further use or characterization. This step is usually accomplished by separation of the amplified nucleic acid fragments by size or by any physical or biochemical means including gel electrophoresis, capillary electrophoresis, chromatography (including sizing, affinity and immunochromatography), density gradient centrifugation and immunoadsorption. An exemplary method is separation of nucleic acid fragments by gel electrophoresis, which provides a rapid and highly reproducible means of sensitive separation of a multitude of nucleic acid fragments, and permits direct, simultaneous comparison of the fragments in several samples of nucleic acids.

[0198] In one embodiment, one or more of the amplified nucleic acid fragments are removed from the gel which was used for identification (see above), according to standard techniques such as chemical extraction, electroelution, or physical excision. The isolated unique nucleic acid fragments may then be inserted into standard vectors, including expression vectors, suitable for transfection or transformation of a variety of prokaryotic (bacterial) or eukaryotic (yeast, plant or animal including human and other mammalian) cells. Alternatively, nucleic acids produced bythe methods may be further characterized, for example by sequencing (i.e., determining the nucleotide sequence of the nucleic acid fragments), by methods described below and others that are standard in the art (see, e.g., U.S. Pat. Nos. 4,962,022 and 5,498,523, which are directed to methods of DNA sequencing). Classical sequencing methods may also be employed such as the Sanger chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) and the Maxam and Gilbert chemical cleavage method (Maxam, A. M. and Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977)).

[0199] In one embodiment, nucleic acids produced by the methods may be further characterized by next generation sequencing. As used herein, the term “next generation sequencing” or “NGS” generally refers to high throughput sequencing technologies, including, but not limited to, massively parallel signature sequencing, high throughput sequencing, sequencing by ligation (e.g., SOLiD sequencing), proton ion semiconductor sequencing, DNA nanoball sequencing, single molecule sequencing, and nanopore sequencing.

[0200] Suitable DNA polymerases for amplification and / or sequencing can include any of those previously disclosed herein. In some embodiments, such polymerase include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp K0D2 (KOD) DNA polymerase, Bacillus sterotherm ophilus (Bst) DNA polymerase, Bacillus caldophilus (Bea) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, Mycobacterium DNA polymerase (Mtb, Mlep), E. coli pol I DNA polymerase, Klenow fragment, T5 DNA polymerase, T7 DNA polymerase, and generally pol I type DNA polymerases; mutants, variants and derivatives thereof, and combinations of the foregoing.

[0201] Suitable nucleic acid polymerases may be mesophilic or thermophilic and are preferably thermophilic and thermostable. As used herein, the term “thermostable nucleic acid polymerase” refers to an enzyme which is relatively stable to heat when compared, for example,to nucleotide polymerases from E. coli and which catalyzes the polymerization of nucleoside triphosphates. Generally, the enzyme initiates synthesis at the 3'-end of the primer annealed to the target sequence and proceeds in the 5'-direction along the template, and if possessing a 5' to 3' nuclease activity, hydrolyzing intervening, annealed probe to release both labeled and unlabeled probe fragments, until synthesis terminates. A representative thermostable enzyme isolated from Thermus aquaticus (Taq) is described in U.S. Pat. No. 4, 889,818 and a method for using it in conventional PCR is described in Saiki et al., 1988, Science 239:487.

[0202] Suitable mesophilic DNA polymerases include Pol I family of DNA polymerases (and their respective Klenow fragments) any of which may be isolated from organism such as E. coli, H. influenzae, D. radiodurans, H. pylori, C. aurantiacus, R. prowazekii, T pallidum, Synechocystis sp., B. subtilis, L. lactis, S. pneumoniae, M. tuberculosis, M. leprae, M. smegmatis, Bacteriophage L5, phi-C31, T7, T3, T5, SP01, SP02, mitochondrial from S. cerevisiae MIP-1, and eukaryotic C. elegans, and D. melanogaster (Astatke, M. et al., 1998, J Mol. Biol. 278, 147-165), pol III type DNA polymerase isolated from any sources, and mutants, derivatives or variants thereof, and the like.[00203J In certain embodiments, the nucleic acid polymerase has 5’->3’ exonuclease activity. As defined herein, “5'— >3' nuclease activity” or “5' to 3' nuclease activity” refers to that activity of a template-specific nucleic acid polymerase including either a 5'— 3 ' exonuclease activity traditionally associated with some DNA polymerases whereby nucleotides are removed from the 5' end of an oligonucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow fragment does not), or a 5'— >3' endonuclease activity wherein cleavage occurs more than one phosphodiester bond (nucleotide) from the 5' end, or both. Taq DNA polymerase has a DNA synthesis-dependent, strand replacement 5'-3' exonuclease activity (see Gelfand, “Taq DNA Polymerase” in PCR Technology: Principles and Applications for DNA Amplification, Erlich, Ed. , Stockton Press, N.Y. (1989), Chapter 2). In some embodiments methods comprising reverse transcription polymerase chain reaction (RT-PCR) are contemplated herein. In such methods, the compositions and reaction mixtures provided herein may further comprise an enzyme that has reverse transcriptase activity. Suitable enzymes having reverse transcriptase activity can be, for example, retroviral reverse transcriptases such as Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, AMV reversetranscriptase, RAV reverse transcriptase, MAV reverse transcriptase, ASLV reverse transcriptases, as well as Lentivirus reverse transcriptases, or corresponding mutants, variants or derivatives thereof having reverse transcriptase activity. As used herein, “mutants, variants, or derivatives” refer to all permutations of a chemical species, which may exist or be produced, that still retains the definitive chemical activity of that chemical species. Some preferred enzymes for use in the invention include those that are RNase H+ enzymes such as, for example, RNase H+ M-MLV or RNase H+ AMV reverse transcriptases. Alternatively, the reverse transcriptases may have reduced, substantially reduced, or eliminated RNase H activity (see, e.g., U.S. Pat. No. 7,078,208, the disclosure of which is fully incorporated by reference in its entirety). RNase H is a processive 5’ and 3’ ribonuclease that is specific for the RNA strand of RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). RNase H activity may be determined by a variety of assays, such as those described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz et al., Nucl. Acids Res. 16:265 (1988) and in Gerard et al., FOCUS 14(5):91 (1992).

[0204] The reverse transcriptase may comprise a mutation as compared to the naturally- occurring reverse transcriptase. For example, 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. Reverse transcriptases which exhibit such increased or decreased functionalities are described in, for example, U.S. Pat. Nos. 7,056,716 and 7,078,208.

[0205] As presented herein, assembled PCR compositions provided herein are effective at reducing PCR run times as compared to an equivalent PCR with a commercially available master mix. This was particularly the case when the nucleic acid sample added to the PCR was a crude lysate and / or the target sequence was present at a low copy number. In some embodiments, this reduction of PCR run times is demonstrated by lower Ct values. In some embodiments, the Ct value is at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, or at least 1000 fold lower when using assembled PCR compositions provided herein compared to an equivalent PCR with a commercially available composition or master mix.

[0206] As used herein the term “Ct” or “Ct value” refers to threshold cycle and signifies the cycle of a PCR amplification assay in which signal from a reporter that is indicative of amplicon generation (e.g., fluorescence) first becomes detectable above a background level. In some embodiments, the threshold cycle or “Ct” is the cycle number at which PCR amplification becomes exponential. In one embodiment, the signal from a reporter, such as fluorescence, is described as delta Rn. As used herein, the term “dRn” or “delta Rn” refers to the difference in the normalized reporter signal (Rn) subtracted from the background signal (baseline) which is then normalized by a passive reference signal. Delta Rn can be determined by the formula Rn+ - Rn-, where Rn+ is the Rn value for a reaction involving all components, including the template, and Rn- is the value for an unreacted sample.

[0207] According to various embodiments, a Ct value may be determined using a derivative of a PCR curve. For example, a first, second, or nth order derivative method may be performed on a PCR curve in order to determine a Ct value. In various embodiments, a characteristic of a derivative may be used in the determination of a Ct value. Such characteristics may include, but are not limited by, a positive inflection of a second derivative, a negative inflection of a second derivative, a zero crossing of the second derivative, or a positive inflection of a first derivative. In various embodiments, a Ct value may be determined using a thresholding and baselining method. For example, 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. From the upper and lower bound 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).

[0208] As used herein the terms “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. In some embodiments, the primary interaction is base specific, e g., A / T and G / C, by Watson / Crick and Hoogsteen-type hydrogen bonding. In some embodiments, base-stacking and hydrophobicinteractions 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. Higgins, eds., IRL Press, Washington, D.C. (1985) and J. Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general, whether such 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. Thus, 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. Further, in general probes and primers of the present teachings are designed to be complementary to a target sequence, such that hybridization of the target and the probes or primers occurs. It will be appreciated, however, that this complementarity need not be perfect; there can be any number of base pair mismatches that interfere with hybridization between the target sequence and the single stranded nucleic acids of the present teachings. However, if the number of base pair mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes or primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions.

[0209] The term “label” as used herein 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

[0210] In some embodiments, provided herein are compositions and reaction systems including such compositions for detection of a target nucleic acid in a crude lysate sample by dPCR. In some embodiments, the compositions and reaction systems include polymerases, deoxynucleoside triphosphates (dNTP), an aqueous extraction buffer, primers, and / or PCRprobes. In some embodiments, the compositions and reaction systems are free of a restriction enzyme.

[0211] In some embodiments, the compositions include one or more polymerases. Such polymerases can be any enzyme capable of replicating a DNA molecule. In some embodiments, 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. In some embodiments, a combination of DNA dependent DNA polymerases and / or a combination of RNA-dependent DNA polymerase can be present in the compositions disclosed herein.

[0212] In some embodiments, the polymerases as used herein are thermostable DNA polymerases. In some embodiments, 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. Preferably a thermostable DNA polymerase does not irreversibly denature at about 90°-100°C under conditions such as is typically required for PCR amplification.

[0213] 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).

[0214] 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.

[0215] In some embodiments, the 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, VENT™ DNA polymerase, DEEPVENT™ 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. 5,912,155), and Tma DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga maritima (see, e.g., U. S. Patent No. 5,374,553, the disclosure of which is incorporated herein by reference in its entirety). Exemplary thermostable polymerases include, but are not limited to, AmpliTaq DNA polymerase and AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific).

[0216] It is to be understood, however, that DNA polymerases from other organisms can also be used herein without departing from the scope or preferred embodiments thereof. As an alternative to isolation, 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.

[0217] In certain embodiments, the concentration of the thermostable DNA polymerase in the composition ad reaction mixtures disclosed herein is about 0.01 to about 500 units per microliter, about 0.01 to about 50 units per microliter, about 0.01 to about 25 units per microliter, about 0.01to about 10 units per microliter, about 0.1 to about 5 units per microliter, about 0.1 to about 2 units per microliter, about 0.1 to about 1 unit per microliter or about 0.1 to about 0.5 units per microliter (units per microliter = U / pL), including all concentrations and concentration ranges within any of the forgoing.

[0218] In some embodiments, the compositions provided herein comprise at least one deoxynucleoside triphosphates (dNTP). In certain embodiments, the composition may further comprise a combination of one or more deoxyribonucleoside triphosphates (dNTPs) and one or more dNTP derivatives. In some embodiments, the composition comprises two to eight different dNTPs and / or dNTP derivatives. In some embodiments, the composition comprises two, three, four, five, or six different dNTPs and / or dNTP derivatives.

[0219] Examples of 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. Examples of possible 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. In certain embodiments, the composition may further comprise a combination of one or more deoxyribonucleoside triphosphates (dNTPs) and one or more dNTP derivatives. In certain embodiments, 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. Such 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).

[0220] The concentration of individual dNTPs, ddNTP, and / or derivatives of each thereof in the composition need not be identical. In some embodiments of the present compositions, 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, orpreferably about 0.2 mM to about 0.8 mM, including any concentrations or range of concentrations within any of the forgoing. In certain embodiments, concentrations of each dNTP and / or ddNTP in the composition is such so that its final concentration in the assembled PCR is about 0.015 mM to about 5 mM, about 0.05 to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.15 mM to about 0.65 mM, about 0.15 mM to about 0.35 mM, or about 0.35 mM to about 0.65 mM, including any concentrations or range of concentrations within any of the forgoing. In some embodiments of the present compositions, dNTP derivatives and / or ddNTP derivatives can be added to give a concentration of each dNTP derivative and / or ddNTP derivative 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. In certain embodiments, concentrations of each dNTP and / or ddNTP in the composition is such so that its final concentration in the assembled PCR is about 0.015 mM to about 5 mM, about 0.05 to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.15 mM to about 0.65 mM, about 0.15 mM to about 0.35 mM, or about 0.35 mM to about 0.65 mM, including any concentrations or range of concentrations within any of the forgoing.

[0221] In certain embodiments, the composition comprises a combination of a dNTP and a corresponding derivative of the same nucleotide thereof, such as a combination of a dNTP and the corresponding alpha-thio-dNTP derivative of the same nucleotide or a combination of a dNTP and the corresponding deaza-dNTP derivative of the same nucleotide. For example, in an embodiment, the composition provided comprises both dGTP and alpha-thio-dGTP. In another embodiment, the composition provided comprises both dGTP and 7-deaza-dGTP. In yet another embodiment, the composition comprises a combination of dATP and 7-deaza-dATP.

[0222] When both a dNTP and a derivative thereof are present in the composition, the relative concentration or concentration ratio of dNTP to derivative may vary. For example, in some embodiments, the relative concentration of dNTP:dNTP derivative is 1 : 1. In some embodiments, the dNTP concentration: dNTP derivative concentration is from about 100: 1 to about 1 : 1, from about 50:1 to about 1.2: 1, from about 25: 1 to about 1.5: 1, from about 10: 1 to about 2: 1. In other embodiments, the dNTP concentration: dNTP derivative concentration is from about 1: 1 to about 1 : 100, from about 1 : 1.2 to about 1 :50, from about 1 :25 to about 1 : 1.5, from about 1 : 110 to about 1 :2. In some embodiments, the dNTP concentration: dNTP derivative concentration is from about2: 1 to about 1 :2. In some embodiments, the dNTP concentratiomdNTP derivative concentration is from about 10: 1 to about 2:1. In some embodiments, the dNTP concentration: dNTP derivative concentration is from about 1 :2 to about 1 : 10. For example, in certain embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 100:1 to about 1.5:1 or from about 50: 1 to about 12:1. In other embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 1 : 1.5 to about 1 : 100 or from about 1 : 12 to about 1 :50. In some embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 2: 1 to about 1 :2. In some embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 10: 1 to about 2: 1. In some embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 1 :2 to about 1: 10. In certain embodiments, the dGTP concentration:7-deaza-dGTP concentration is from about 100: 1 to about 1.5: 1 or from about 50: 1 to about 12: 1. In other embodiments, the dGTP concentration:7-deaza-dGTP concentration is from about 1 : 1.5 to about 1 : 100 or from about 1 :12 to about 1 :50. In some embodiments, the dGTP concentration: 7-deaza- dGTP concentration is from about 2: 1 to about 1 :2. In some embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 10: 1 to about 2: 1. In some embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 1:2 to about 1 : 10.

[0223] In some embodiments, the compositions comprise one or more aqueous extraction buffers. In some embodiments, 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”.

[0224] In some embodiments, 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.

[0225] In some embodiments, 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. Such primers include, but are notlimited to, target-specific primers (which are preferably gene-specific primers), oligo(dT) primers, random primers or arbitrary primers. Additional primers that can be used for amplification of the DNA molecules according to the methods disclosed herein will be apparent to one of ordinary skill in the art. It is to be understood that a vast array of primers can be useful in the present compositions, methods and kits, including those not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.[00226J In some embodiments, the final concentration of primers can range from about 25 nM to about 2000 nM, such as about 50 nM to about 1700 nM, about 75 nM to about 1500 nM, about 100 nM to about 1200 nM, about 200 nM to about 1000 nM, or any range in between. In some exemplary embodiments, the concentration of each primer is between about 400 nM to about 900 nM, including all amounts or ranges in between.

[0227] In accordance with the present teachings, the compositions can further comprise probes for the detection of target nucleic acids. Various probes are known in the art, for example (TaqMan™ probes (see, e.g., U.S. Pat. No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99 / 21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise™ / Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes (see, e.g., Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (see, e.g., U.S. Pat. No. 6,590,091), pseudo knot probes (see, e.g., U.S. Pat. No. 6,589,250), cyclicons (see, e.g., U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (see, e.g., U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes, 14:321-328; Svanvik et al., 2000, Anal Biochem. 281 :26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Res., 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Res., 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc., 124:9606-9612; Broude et al., 2002, Trends Biotechnol., 20:249-56; Huang et al., 2002, Chem Res. Toxicol., 15: 118-126; and Yu et al., 2001, J. Am.Chem. Soc., 14: 11 155-11 161. Probes can comprise reporter dyes such as, for example, 6- carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET). Detector probes can also comprise quencher moieties such as tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Thermo Fisher Scientific), and Dabsyl and Dabcel sulfonate / carboxylate Quenchers (Epoch). Probes can also comprise two probes, wherein for example a fluor is on one probe, and a quencher on the other, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on a target alters the signal signature via a change in fluorescence.

[0228] 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. Suitable detectable labels may include, for example, fluoresceins (e.g., 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 6-HAT; 6- JOE; 6-carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY® fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-m ethylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g., calcium crimson, calcium green, calcium orange, cal cofluor white), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., GFP, EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, BODIPY® FL / BODIPY® FL, Fluorescein / QSY7 and QSY9), LysoTracker and LysoSensor (e g., LysoTracker Blue DND-22, LysoTracker Blue- White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND- 153, LysoSensor Yellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran),Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, 5-ROX (carboxy- X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in, e.g., US Publication No. 2009 / 0197254), among others as would be known to those of skill in the art.[00229J In some embodiments, the probes are designed according to the methods and principles described in, for example, U.S. Patent No. 6,727,356 (the disclosure of which is incorporated herein by reference in its entirety). Some probes can be sequence-based, for example 5' nuclease probes and some, such as SYBR® Green can be non-sequence specific DNA-binding dyes. In some preferred embodiments, the detector probe is a TaqMan™ probe (Applied Biosystems, Foster City, CA). It is to be understood that a wide variety of probes are known in the art that can be used in the present compositions, methods and kits, including those not specifically disclosed herein.

[0230] In some embodiments, the probe concentration in a working solution can range from about 5 nM to about 750 nM, such as about 10 nM to about 600 nM, about 25 nM to about 500 nM, about 50 nM to about 400 nM, about 75 nM to about 300 nM, or any number in between. In some exemplary embodiments, the concentration of each probe is between about 100 nM to about 250 nM, including all amounts or ranges in between.

[0231] The compositions may also comprise one or more PCR inhibitor blocking agents. Such PCR inhibitor blocking agents can be added to the compositions or reaction mixtures disclosed herein to assist in overcoming the inhibition of PCR reactions by a variety of compounds often found in biological samples used for nucleic acid preparation, isolation, or purification. In some embodiments, the PCR inhibitor blocking agent(s) can reduce the amount of PCR inhibition by a PCR inhibitor or inhibitors from some percentage above zero up to 100% compared to the level of inhibition observed in the absence of such PCR inhibitor blocking agents. For example, inhibition can be reduced by at least about 0.5%, about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 90%, about 95% or about 100% or any percentage in between.

[0232] In some embodiments, the PCR inhibitor blocking agent is a protein. In some embodiments, such proteins can include, but are not limited to, albumins, gelatins, and DNA- binding proteins, or peptide or polypeptide variants, fragments or derivatives thereof. In some embodiments, other non-protein based PCR inhibitor blocking agents for use in the present teachings can include, for example, deferoxamine mesylate. In some embodiments, the compositions can comprise a combination of PCR inhibitor blocking agents and / or proteins. For example, in some embodiments the compositions comprise an albumin, a gelatin, or a combination of albumin and gelatin. In some embodiments, the albumin or gelatin may be selected from serum albumin, fish gelatin, or a combination of serum albumin and fish gelatin. The serum albumin can be from any animal, e.g., bovine serum albumin (BSA), human serum albumin (HSA). In some embodiments, the albumin is derived from other species of animals which are well-known to those of skill in the art. In some embodiments the albumin is a recombinant albumin, such as recombinant BSA (rBSA). The gelatin can be from any animal, e.g., fish gelatin, bovine gelatin. In some embodiments, the gelatin is derived from other species of animals which are well-known to those of skill in the art. In some embodiments the gelatin is a recombinant gelatin, such as recombinant human gelatin. In some embodiments, the DNA- binding proteins can include, but are not limited to T4 gene 32 protein (T4 gp32).

[0233] Certain PCR inhibitor blocking compounds or agents can be added to the present compositions to give a concentration in the composition of about .0001 mg / mL to about 10 mg / mL, about .001 mg / mL to about 8 mg / mL, about 0.01 mg / mL to about 6 mg / mL, about 0.05 mg / mL to about 4 mg / mL, about 0.1 mg / mL to about 3 mg / mL or about 0.5 mg / mL to about 2 mg / mL, including any concentrations or range of concentrations within any of the forgoing. 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. In some embodiments, the compositions as described herein comprise a PCR inhibitor blocking protein, such as albumin, at a concentration such that its concentration in an assembled PCR is about 0.001 mg / mL to about 10.0 mg / mL, 0.005 mg / mL to about 5.0 mg / mL, about 0.01 mg / mL to about 4.0 mg / mL, about 0.05 mg / mL to about 3.0 mg / mL or about 0.1 mg / mL to about 2.0 mg / mL, including any concentrations or range of concentrations within any of the forgoing. In another embodiment, the compositions asdescribed herein comprise a PCR inhibitor blocking protein, such as gelatin at a concentration such that its concentration in an assembled PCR is about 0.005% (w / v) to about 2% (w / v), about 0.01% (w / v) to about 1.0% (w / v), and more specifically about 0.05% (w / v) to about 0.5% (w / v), including any concentrations or range of concentrations within any of the forgoing. In yet another embodiment, the compositions as described herein comprise a combination of albumin and gelatin at any of the foregoing concentrations. In some preferred embodiments, the albumin is at about 0.05mg / mL to 5 mg / mL and gelatin is at a concentration of about 0.01% (w / v) to about 1%. In some embodiments, the albumin is bovine serum albumin (BSA), and the gelatin is a fish gelatin and / or a bovine gelatin.

[0234] In some embodiments, the composition can comprise one, two, three, four, five or more different PCR inhibitor blocking proteins and / or agents. For example, in some embodiments the composition comprises an albumin, a fish gelatin and a bovine gelatin. In some embodiments, 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. In some embodiments, one or more PCR inhibitor blocking agents are added to the compositions or reaction mixtures to help to overcome inhibition of PCR by a variety of inhibitors often found in biological samples, Such inhibitors include, for example, heparin (blood); hematin (blood); EDTA (blood); citrate (blood); immunoglobin G (blood, serum); humic acid (soil, feces); lactoferrin (milk, saliva, other secretory fluids); urea (urine); plant polysaccharides (plants); melanin (skin, hair); myoglobin (tissue); and indigo dye (textiles). The addition of PCR inhibitor blocking agents, both individually and in combination, can increase tolerance to such PCR inhibitor contaminants. Thus, the present compositions can further comprise agents that work alone or in combination to increase tolerance to various PCR inhibitors including, for example, humic acid, hematin, and heparin. In some embodiments each PCR inhibitor blocking agent or protein in the composition or reaction mixture can block a different inhibitor or group of inhibitors than the other PCR inhibitor blocking agent or protein in the composition or reaction mixture. In some embodiments, the different PCR inhibitor blocking agents or proteins in the composition or reaction mixture can block the same inhibitor or group of inhibitors. In some embodiments, the different PCR inhibitor blocking agents or proteins in the composition or reaction mixture can block an overlapping number of inhibitor of a group of inhibitors. For example, in some embodiments, gelatin is effective at reducing PCR inhibition by at least humicacid and heparin, and albumin is effective at reducing PCR inhibition by at least humic acid and hematin.

[0235] In some embodiments, one or more additional components and / or additives can be incorporated in the present compositions, methods, and kits to optimize the synthesis of nucleic acids from a nucleic acid template. In some embodiments, the composition can comprise additional components and / or additives which are capable of facilitating or enhancing nucleic acid synthesis reactions (e.g., reagents for facilitating or enhancing PCR). Components and / or additives which enhance nucleic acid synthesis can be organic or inorganic compounds. Some components and / or additives useful in the present compositions, methods, and kits include polypeptides as well as nonpolypeptide components. Such components and / or additives can include, but are not limited to, for example, single-stranded binding DNA-binding (SSB) proteins, sulfur-containing compounds, acetate-containing compounds, dimethylsulfoxide (DMSO), glycerol, formamide, betaine, tetramethylammonium chloride (TMAC), ectoine, sodium azide, kathon, polyols, NaNs, buffers, surfactants, detergents (e g., TWEEN 20, NP-40, Triton X-100, and CHAPS), a component or compound used for hot start PCR, a passive reference control to minimize sample-to-sample and / or well-to-well variations in quantitative real-time DNA-detection assays, and / or uracil DNA glycosylase, to name just a few. The composition may further comprise crowding agents such as Ficoll 70, glycogen, and polyethylene glycol (PEG). Those of ordinary skill in the art will be able to identify additional reagents and / or additives for use in accordance with the present compositions, methods and kits.

[0236] In some embodiments, the compositions include a buffer component, such as a buffered salt solution. In some embodiments, the buffer agent provides appropriate pH conditions to maintain stability of the DNA polymerase enzyme. The terms "stable" and "stability" as used herein generally mean the retention by a composition, such as an enzyme composition, of at least 70%, preferably at least 80%, and most preferably at least 90%, of the original enzymatic activity (in units) after the enzyme or composition containing the enzyme has been stored for about 3 days at a temperature of about room temperature (e.g., about 20°C to about 25°C), about one to eight weeks at a temperature of about 4°C, about two to six months at a temperature of about - 20°C, and about six months or longer at a temperature of about -80 °C. Examples of such buffering agents can include, for example, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES TAPS, PIPES, and CAPS. In some embodiments, the buffer concentration of thecompositions 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.

[0237] Examples of 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. In certain embodiments, the composition comprises more than one (e.g., two, three, four, five, six, etc.) salt component. For example, the compositions described herein can comprise two different salts. In other embodiments, the compositions comprise three different salts. In still other embodiments, the compositions comprise four different salts. In one embodiment, 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. Without limitation, in certain embodiments where 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. Without limitation, in certain embodiments where 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. It is to beunderstood that 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.

[0238] In some embodiments, the compositions comprise a passive reference control component. In some embodiments, 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. In an embodiment, a reference chromophore, specifically a fluorophore, is included as the passive reference control. In an embodiment, the reference chromophore is the fluorescent dye. In some embodiments, the fluorescent dye is a ROX dye (Thermo Fisher Scientific). In some embodiments, the fluorescent dye is a MUSTANG PURPLE dye (Thermo Fisher Scientific). The passive reference can be included in the composition at a concentration such that its concentration in the assembled PCR is about 10 to about 750 nM or about 20 nM to about 500 nM, or about 50 nM to about 200 nM, including any concentration falling within the forgoing ranges.

[0239] In some embodiments, uracil DNA glycosylase (UNG or UDG) can be included in the compositions or kits provided herein. The enzyme is commercially available from a number of commercial sources, for example Thermo Fisher Scientific, Enzymatics, New England Biolabs, Genscript, or USB. In some embodiments, UNG is thermolabile. In other embodiments, UNG is thermostable. Thermolabile or thermostable UNG can be included in the composition at a concentration such that its concentration in the final assembled PCR is about 0.0001 U / pL to about 50 U / pL, about 0.0005 U / pL to about 10 U / pL, about 0.001 U / pL to about 5 U / pL, 0.005 U / pL to about 1.0 U / pL, or 0.01 U / pL to about 0.5 U / pL (U / pL = units per microliter), including any concentration or range falling within the forgoing ranges.

[0240] Undesired amplification reactions, which can occur during the PCR process, usually begin during assembly of the reaction mixtures, or while the thermal cycler is heating to the initial denaturation temperature. These spurious reactions can be minimized by performing "hot start" amplification. In general, hot start techniques limit the availability of an essential reaction component until an elevated temperature, often >60°C, is reached.

[0241] In some embodiments, the compositions are provided as a concentrated stock solution or mix. As used herein, the term "concentrated stock" means at a concentration that requiresfurther dilution in order to achieve optimal concentration for use in a solution to perform a particular function (such as PCR amplification). For example, compositions may be stock solutions of about 2X, about 3X, about 4X, about 5X, about 6X, about 10X, and so on, including any amount between any of the forgoing. In some embodiments, the compositions may require greater than 2X, greater than 3X, greater than 4X, greater than 5X, greater than 6X, greater than 10X, and so on, including any amount between any of the forgoing, dilution to be at working, or optimal, concentration for use, for example, in nucleic acid synthesis or amplification methods. In some embodiments, the compositions described herein may also be provided at a working concentration or as a “working solution or mix.” As used herein “working concentration” or “working solution or mix” is used to refer to a solution or mix that is at or near the optimal concentration used to perform a particular function or reaction (such as amplification or PCR). In some embodiments, a composition that is at a working concentration needs no or minimal dilution prior to use. For example, in some embodiments a compositions that is at a working concentration is at about a IX, about a 1.25X, about a 1.5X, about a 1.8X, about a 2X, or about a 2.2X concentration of the final concentration in the assembled reaction mixture.

[0242] In some embodiments, the compositions are formulated as master mixes. In some embodiments, the master mix is an RNA master mix. Master mixes can improve efficiency and reduce errors associated with the assembly of large number of reactions required for high- throughput analysis. In some embodiments, master mixes can contain combination of reagents common to all reactions. For example, in some embodiments the master mix can contain a buffer, a salt, such as MgCh, deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, a detergent, and a PCR inhibitor blocking agent. For use in certain assays, each reaction would then contain an aliquot of the common master mix and a specific target nucleic acid template and at least one primer. In some embodiments, master mixes can be manufactured and distributed as a concentrated stock solution or mixture. The master mix can then be diluted when final reactions are assembled. In other embodiments, the master mix can be manufactured and distributed as a working solution or mixture.

[0243] In some embodiments, the compositions are reaction mixtures. As used herein “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.

[0244] In some embodiments, the dNTP of the reaction mixture is selected from dGTP, dCTP, dATP and dTTP. In some embodiments, 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. In some embodiments, the reaction mixture comprises a dNTP / dNTP derivative blend comprising some combination of the aforementioned dNTPs and dNTP derivatives. In some embodiments, the nucleic acid template of the reaction mixture is DNA. In some embodiments, the DNA in the reaction mixture is genomic DNA (gDNA) or complementary DNA (cDNA).

[0245] In some embodiments, 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. In some embodiments, 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.

[0246] In some embodiments, 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.Kits

[0247] In some embodiments, provided herein are kits including compositions or reaction systems for detection of a target nucleic acid in a crude lysate sample by dPCR. In some embodiments, the kits comprise the compositions or reaction systems or packaged containers comprising the compositions or reaction systems. In some embodiments, the kits are free of a restriction enzyme. In some embodiments, the kits are free of a phenol -chloroform extraction composition.

[0248] In some embodiments, the compositions or reaction systems can be assembled into kits for use in nucleic acid synthesis or amplification reactions. The kits can further comprise additional reagents used in one or more assays used to synthesize, detect or quantify nucleicacids. In one embodiment, the composition or reaction systems may be present in a kit for use in the amplification of a nucleic acid molecule, such as in PCR.

[0249] In some embodiments, the kits comprise a carrier, such as a box, carton, tube, or the like, having in close confinement therein, a container, such as a vial, tube, ampule, plate, bottle and the like. When more than one enzyme is included in a kit (for example, a DNA polymerase and a reverse transcriptase or a DNA polymerase and UNG), the enzymes may be in a single container as mixtures of two or more enzymes (e.g., 2, 3, 4, 5, etc.), or in separate containers.

[0250] In some embodiments, the kits comprise master mix compositions for use in nucleic acid synthesis or amplification reactions. Such compositions may be formulated as concentrated stock (e.g., 2X, 3X, 4X, 5X, 6X, etc.). In some embodiments, the compositions can be formulated as concentrated stock in a single tube or container. In some embodiments, such concentrated stock compositions may further comprise a combination of PCR inhibitor blocking agents, a non-ionic detergent other than TWEEN 20, a hot start component, and / or a passive reference dye in a buffered solution. In some additional embodiments, such buffer solutions may comprise glycerol, DMSO, and / or a second salt.

[0251] In some embodiments, 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. For example, the probe can be a TaqMan™ probe, a HydrolEasy™ probe, a minor groove binding (MGB) probe, a locked nucleic acid (LNA) probe, a SYBR Green or SYBR GreenER™ probe, or a cycling probe technology (CPT) probe.

[0252] In some embodiments, the kit further comprises a control nucleic acid sample, and at least one primer pair specific for PCR amplification of a nucleic acid target in the control nucleic acid sample, and / or at least one probe specific for the amplified nucleic acid target in the control nucleic acid sample. A probe for detecting the amplification can also be included in the kit.

[0253] Components of the kit other than the composition may be provided in individual containers or in a single container, as appropriate. In some embodiments, instructions, manuals, and / or protocols for using the kit advantageously can be provided. Many assays are suitable for use in synthesizing target nucleic acids using the disclosed compositions and reaction mixtures and are contemplated herein as would be understood by one of skill in the art.Examples

[0254] The invention is further described in the context of the following examples which are presented by way of illustration, not of limitation.Example 1 : dPCR with and without restriction enzyme incubation

[0255] A grapevine was infected with X. fastidiosa. Plant tissue was collected from the infected grapevines at four weeks and eight weeks after infection.

[0256] A set of purified samples was prepared by extracting nucleic acids from the collected plant tissue of the infected grapevine in a phenol -chloroform extraction composition. The extracted sample was then subjected to a conventional qPCR-based workflow that included a 30- minute restriction enzyme incubation step at 35 °C to purify the samples. The resulting purified sample was collected and run on a QuantStudio™ Absolute Q™ Digital PCR System (Thermo Fisher Scientific).

[0257] A set of crude lysate samples was prepared by grinding the collected plant tissue of the infected grapevines in a GES-PVP extraction buffer. Each sample was then centrifuged, and the supernatant was collected. The collected supernatant was prepared, loaded onto a plate, and run on a QuantStudio™ Absolute Q™ Digital PCR System without the conventional restriction enzyme incubation step or a purification step used to generate the purified samples.

[0258] FIG. 1A-1D show the resulting variability charts and FIG. 2A-FIG. 3D show the resulting copy number detection levels of the same dPCR assays.

[0259] In a first sample tested at four weeks, 14.4 cp / pL of the target nucleic acid was detected without the conventional restriction enzyme incubation step and 16.0 cp / pL was detected with the conventional restriction enzyme incubation step (FIG. 1A, FIG. 2A, and FIG. 2B).

[0260] In a second sample tested at four weeks, 108.4 cp / pL of the target nucleic acid was detected without the conventional restriction enzyme incubation step and 99.8 cp / pL was detected with the conventional restriction enzyme incubation step (FIG. IB, FIG. 2C, and FIG. 2D). In a first sample tested at eight weeks, 343.1 cp / pL of the target nucleic acid was detected without the conventional restriction enzyme incubation step and 346.6 cp / pL was detected with the conventional restriction enzyme incubation step (FIG. 1C, FIG. 3 A, and FIG. 3B).

[0261] In a second sample tested at eight weeks, 683.8 cp / pL of the target nucleic acid was detected without the conventional restriction enzyme incubation step and 621.0 cp / pL was detected with the conventional restriction enzyme incubation step (FIG. ID, FIG. 3C, and FIG. 3D).

[0262] The Absolute Q™ Digital PCR System detected X. fastidiosa from asymptomatic infected grapevines at 4 weeks post infection in crude lysate samples without full DNA extraction and at similar levels to full DNA extraction, which eliminates the need for the full DNA extraction.Example 2: dPCR versus qPCR

[0263] Purified samples from grapevine plant tissue that was collected at 4-week and 8-week after infected with A. fastidiosa were also tested using quantitative PCR (qPCR) However, in the qPCR workflow, infection was not detected 4 weeks post-infection with purified samples. The qPCR only detected infection 8 weeks post-infection with purified samples. In contrast the dPCR system consistently detected X. fastidiosa from grape crude lysate at as early as 4 weeks post infection (asymptomatic) without a preliminary purification step.

[0264] The same samples infected with X. fastidiosa in crude grapevines were tested using both dPCR and qPCR, both with a purification step. The resulting data is shown in Table 1 below and FIG. 4.Table 1 : Crude Extraction

[0265] As shown in FIG. 4, the R-square value was 0.97, meaning a statistically significant correlation was established between the two sets of data.

[0266] The Absolute Q™ Digital PCR workflow was both faster and less expensive without the sample purification step, due primarily to the decreased labor costs. Also, the ability of dPCR to detect infections early is advantageous because individuals gain advantage by acting before physical symptoms are present. This allows for early isolation of infected hosts and “culling” with more precision. The comparative experiment demonstrated a statistically significant similarity between dPCR and purified qPCR data. This advance highlights the potential of the methods disclosed herein in addressing disease detection and monitoring challenges in plant biology.

[0267] A first aspect 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.

[0268] In the first aspect, 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.

[0269] In the first aspect, the method is performed without incubating the crude lysate sample with a restriction enzyme.

[0270] In the first aspect, the extraction buffer is an aqueous extraction buffer.

[0271] In the first aspect, the extraction buffer is a lysis buffer.

[0272] In the first aspect, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0273] In the first aspect, 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.

[0274] In the first aspect, the reaction mixture is an unpurified reaction mixture.

[0275] In the first aspect, the crude lysate sample is an unpurified lysate sample.

[0276] In the first aspect, 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.

[0277] In the first aspect, 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.

[0278] In the first aspect, 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.

[0279] In the first aspect, the microfluidic array plate is a single plate.

[0280] In the first aspect, the method further comprises quantifying an amount of the target nucleic acid based on the fluorescence detected.

[0281] In the first aspect, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0282] In the first aspect, 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.

[0283] In the first aspect, 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.

[0284] In the first aspect, 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.

[0285] In the first aspect, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0286] In a second aspect, a method of detecting pathogen in a subject, comprises performing an amplification reaction on a reaction mixture including a crude lysate sample to generate amplicons of a target nucleic acid of the pathogen, wherein the crude lysate sample includesbiological tissue from the subject; and detecting fluorescence from a probe hybridized to the amplicons to determine if the pathogen is present in the subject.

[0287] In the second aspect, the presence of the pathogen is determined before the subject is symptomatic of an infection by the pathogen.

[0288] In the second aspect, 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.

[0289] In the second aspect, the method is performed without incubating the crude biological sample or the crude lysate sample with a restriction enzyme.

[0290] In the second aspect, the extraction buffer is an aqueous extraction buffer.

[0291] In the second aspect, the extraction buffer is a lysis buffer.

[0292] In the second aspect, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0293] In the second aspect, 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.

[0294] In the second aspect, the reaction mixture is an unpurified reaction mixture.

[0295] In the second aspect, the crude lysate sample is an unpurified biological sample.

[0296] In the second aspect, 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.

[0297] In the second aspect, 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.

[0298] In the second aspect, 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.

[0299] In the second aspect, the microfluidic array plate is a single plate.

[0300] In the second aspect, the method further comprises quantifying an amount of the target nucleic acid based on the fluorescence detected.

[0301] In the second aspect, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0302] In the second aspect, 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.

[0303] In the second aspect, 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.

[0304] In the second aspect, 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.

[0305] In the second aspect, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0306] In a third aspect, a polymerase chain reaction (PCR) system, comprises a reaction mixture including a crude lysate sample, at least one probe configured to hybridize to amplicons of a 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 add the at least one deoxynucleoside triphosphate (dNTP) for synthesizing the amplicons, wherein the PCR system is used to perform an amplification reaction on the reaction mixture including the crude lysate sample to generate the amplicons of the target nucleic acid, and wherein the crude lysate sample includes biological tissue.

[0307] In the third aspect, 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.

[0308] In the third aspect, the reaction mixture further includes a reverse transcriptase.

[0309] In the third aspect, the target nucleic acid belongs to a pathogen infecting a host or subject.

[0310] In the third aspect, presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

[0311] In the third aspect, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0312] In the third aspect, the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

[0313] In the third aspect, 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.

[0314] In the third aspect, the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

[0315] In the third aspect, the extraction buffer is a lysis buffer.

[0316] In the third aspect, the extraction buffer is an aqueous extraction buffer.

[0317] In the third aspect, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0318] In the third aspect, the reaction mixture is an unpurified reaction mixture, and / or the crude lysate sample is an unpurified biological sample.

[0319] In the third aspect, 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.

[0320] In the third aspect, the amplification reaction is performed without extracting the crude lysate sample with a chloroform -based compound and / or a phenol-based compound.

[0321] In the third aspect, the amplification reaction is performed on a microfluidic array plate of a digital PCR instrument.

[0322] In the third aspect, the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

[0323] In the third aspect, the microfluidic array plate is a single plate.

[0324] In the third aspect, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0325] In the third aspect, 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.

[0326] In the third aspect, 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.

[0327] In the third aspect, 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.

[0328] In a fourth aspect, 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.

[0329] In the fourth aspect, 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.

[0330] In the fourth aspect, the reaction mixture further includes a reverse transcriptase.

[0331] In the fourth aspect, the target nucleic acid belongs to a pathogen infecting a host or subject.

[0332] In the fourth aspect, presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

[0333] In the fourth aspect, the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

[0334] In the fourth aspect, the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

[0335] In the fourth aspect, 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.

[0336] In the fourth aspect, the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

[0337] In the fourth aspect, the extraction buffer is a lysis buffer.

[0338] In the fourth aspect, the extraction buffer is an aqueous extraction buffer.

[0339] In the fourth aspect, the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

[0340] In the fourth aspect, wherein the second mixture is mixed with the crude biological sample to produce a reaction mixture.

[0341] In the fourth aspect, the reaction mixture is an unpurified reaction mixture.

[0342] In the fourth aspect, the crude lysate sample is an unpurified biological sample.

[0343] In the fourth aspect, 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.

[0344] In the fourth aspect, 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.

[0345] In the fourth aspect, the target nucleic acid is amplified on a microfluidic array plate of a digital PCR instrument.

[0346] In the fourth aspect, the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

[0347] In the fourth aspect, the microfl uidic array plate is a single plate.

[0348] In the fourth aspect, the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

[0349] In the fourth aspect, 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.

[0350] In the fourth aspect, 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.

[0351] In the fourth aspect, 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.

[0352] All above-mentioned references are hereby incorporated by reference herein in their entireties.

[0353] The foregoing is illustrative of embodiments and is not to be construed as limiting thereof. Although a few embodiments have been described, those skilled in the art will readily appreciate that many modifications are possible in embodiments without materially departing from the novel teachings. Accordingly, all such modifications are intended to be included within the scope of the disclosure as defined in the claims. Therefore, it is to be understood that the foregoing is illustrative of various embodiments and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. 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.

2. The method of claim 1, further comprising: 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.

3. The method of any one of claims 1 or 2 being performed without incubating the crude lysate sample with a restriction enzyme.

4. The method of any one claims 2 or 3, wherein the extraction buffer is an aqueous extraction buffer.

5. The method of any one of claims 2-4, wherein the extraction buffer is a lysis buffer.

6. The method of any one of claims 2-5, wherein the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

7. The method of any one of claims 2-6, wherein 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.

8. The method of any one claims 1-7, wherein the reaction mixture is an unpurified reaction mixture.

9. The method of any one of claims 1-7, wherein the crude lysate sample is an unpurified lysate sample.

10. The method of any one of claims 1-9, wherein 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.

11. The method of any one of claims 1-10, wherein 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.

12. The method of claim 11, wherein 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.

13. The method of any one of claims 11 or 12, wherein the microfluidic array plate is a single plate.

14. The method of any one of claims 1-13, further comprising: quantifying an amount of the target nucleic acid based on the fluorescence detected.

15. The method of any one of claims 1-14, wherein the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

16. The method of any one of claims 1-15, wherein 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.

17. The method of any one of claims 1 -15, wherein 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.

18. The method of claim 1-17, wherein 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.

19. The method of claim 1-17, wherein the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

20. A method of detecting pathogen in a subject, comprising: performing an amplification reaction on a reaction mixture including a crude lysate sample to generate amplicons of a target nucleic acid of the pathogen, wherein the crude lysate sample includes biological tissue from the subject; and detecting fluorescence from a probe hybridized to the amplicons to determine if the pathogen is present in the subject.

21. The method of claim 20, wherein the presence of the pathogen is determined before the subject is symptomatic of an infection by the pathogen.

22. The method of any one of claims 20 and 21, further comprising: 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.

23. The method of any one of claims 20-22 being performed without incubating the crude biological sample or the crude lysate sample with a restriction enzyme.

24. The method of any one of claims 22 or 23, wherein the extraction buffer is an aqueous extraction buffer.

25. The method of any one of claims 22-24, wherein the extraction buffer is a lysis buffer.

26. The method of any one of claims 22-25, wherein the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

27. The method of any one of claims 22-26, wherein 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.

28. The method of any one of claims 20-27, wherein the reaction mixture is an unpurified reaction mixture.

29. The method of any one of claims 20-27, wherein the crude lysate sample is an unpurified biological sample.

30. The method of any one of claims 20-29, wherein 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.

31. The method of any one of claims 20-30, wherein 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.

32. The method of claim 31, wherein 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, andthe performing of the amplification reaction includes thermocycling the reaction mixture in the plurality of microchambers.

33. The method of any one of claims 31 or 32, wherein the microfluidic array plate is a single plate.

34. The method of any one of claims 20-33, further comprising: quantifying an amount of the target nucleic acid based on the fluorescence detected.

35. The method of any one of claims 20-34, wherein the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

36. The method of any one of claims 20-35, wherein 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.

37. The method of any one of claims 20-35, wherein 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.

38. The method of claim 20-37, wherein 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.

39. The method of claim 20-37, wherein the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

40. A polymerase chain reaction (PCR) system, comprising: a reaction mixture including a crude lysate sample, at least one probe configured to hybridize to amplicons of a 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 add the at least one deoxynucleoside triphosphate (dNTP) for synthesizing the amplicons, wherein the PCR system is used to perform an amplification reaction on the reaction mixture including the crude lysate sample to generate the amplicons of the target nucleic acid, and wherein the crude lysate sample includes biological tissue.

41. The PCR system of claim 40, wherein 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.

42. The PCR system of any one of claims 40 or 41, wherein the reaction mixture further includes a reverse transcriptase.

43. The PCR system of any one of claims 40-42, wherein the target nucleic acid belongs to a pathogen infecting a host or subject.

44. The PCR system of claim 43, wherein presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

45. The PCR system of any one of claims 43 or 44, wherein the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

46. The PCR system of any one of claims 40-45, wherein the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

47. The PCR system of any one of claims 40-46, wherein 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.

48. The PCR system of claim 47, wherein the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

49. The PCR system of claim 47, wherein the extraction buffer is a lysis buffer.

50. The PCR system of any one of claims 47 or 49, wherein the extraction buffer is an aqueous extraction buffer.

51. The PCR system of claim 50, wherein the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

52. The PCR system of any one of claims 40-51, wherein the reaction mixture is an unpurified reaction mixture, and / or the crude lysate sample is an unpurified biological sample.

53. The PCR system of any one of claims 40-52, wherein the crude lysate sample is prepared without extracting the crude biological sample or the crude lysate sample with a chloroformbased compound and / or a phenol-based compound.

54. The PCR system of any one of claims 40-53, wherein the amplification reaction is performed without extracting the crude lysate sample with a chloroform -based compound and / or a phenol-based compound.

55. The PCR system of any one of claims 40-54, wherein the amplification reaction is performed on a microfluidic array plate of a digital PCR instrument.

56. The PCR system of claim 55, wherein the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

57. The PCR system of any one of claims 55 or 56, wherein the microfluidic array plate is a single plate.

58. The PCR system of any one of claims 40-57, wherein the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

59. The PCR system of any one of claims 40-58, wherein 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.

60. The PCR system of any one of claims 40-58, wherein 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.61 . The PCR system of any one of claims 40-60, wherein 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.

62. A digital polymerase chain reaction (PCR) kit, comprising: 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.

63. The PCR kit of claim 62, wherein 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.

64. The PCR kit of any one of claims 62 or 63, wherein the reaction mixture further includes a reverse transcriptase.

65. The PCR kit of any one of claims 62-64, wherein the target nucleic acid belongs to a pathogen infecting a host or subject.

66. The PCR kit of claim 65, wherein presence of the pathogen is determined before the host or subject is symptomatic of an infection by the pathogen.

67. The PCR kit of any one of claims 65 or 66, wherein the pathogen is one selected from a bacterium, a virus, a fungus, a protozoan, a prion, a viroid, and a disease-causing parasite.

68. The PCR kit of any one of claims 62-67, wherein the crude lysate sample is prepared without incubating the crude lysate sample with a restriction enzyme.

69. The PCR kit of any one of claims 62-68, wherein 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.

70. The PCR kit of any one of claims 62-69, wherein the crude lysate sample is prepared without incubating the crude biological sample with a restriction enzyme.

71. The PCR kit of any one of claims 62-70, wherein the extraction buffer is a lysis buffer.

72. The PCR kit of any one of claims 62-71, wherein the extraction buffer is an aqueous extraction buffer.

73. The PCR kit of claim 72, wherein the aqueous extraction buffer comprises guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium lauroyl sarcosinate, and polyvinylpyrrolidone.

74. The PCR kit of any one of claims 62-73, wherein the second mixture is mixed with the crude biological sample to produce a reaction mixture.

75. The PCR kit of claim 74, wherein the reaction mixture is an unpurified reaction mixture.

76. The PCR kit of any one of claims 62-75, wherein the crude lysate sample is an unpurified biological sample.

77. The PCR kit of any one of claims 62-76, wherein the crude lysate sample is prepared without extracting the crude biological sample or the crude lysate sample with a chloroformbased compound and / or a phenol-based compound.

78. The PCR kit of any one of claims 62-77, wherein the target nucleic acid in the crude lysate sample is amplified without extracting the crude biological sample or the crude lysate sample with a chloroform -based compound and / or a phenol-based compound.

79. The PCR kit of any one of claims 62-78, wherein the target nucleic acid is amplified on a microfluidic array plate of a digital PCR instrument.

80. The PCR kit of claim 79, wherein the microfluidic array plate includes a plurality of microchambers in which a portion of the reaction mixture including the crude lysate sample is stored.

81. The PCR kit of any one of claims 79 or 80, wherein the microfluidic array plate is a single plate.

82. The PCR kit of any one of claims 62-81, wherein the biological tissue is from one selected from at least one plant, at least one human, and at least one animal.

83. The PCR kit of any one of claims 62-82, wherein 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.

84. The PCR kit of any one of claims 62-82, wherein 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.

85. The PCR kit of any one of claims 62-84, wherein 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.

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