Method for sample preparation

Abstract

Methods for preparing a sample containing nucleic acids are provided herein. In some embodiments, the method comprises heating the sample above its boiling point in a closed heating chamber to rapidly extract nucleic acids from biological entities present in the sample; and analyzing the nucleic acids from the heat-treated sample. In some embodiments, the treated sample may contain one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 63 / 348,750, filed on June 3, 2022, which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Background Two methods for nucleic acid extraction and purification are mainly used during sample preparation for nucleic acid analysis. The first method involves purification of nucleic acids from other molecular species present in the sample by extraction. Such methods often include phenol / chloroform extraction followed by ethanol precipitation, use of chaotropic lysis agents followed by ion - exchange resins, or use of silica resins. These methods provide high - quality nucleic acids (e.g., high - purity nucleic acids), but are time - consuming and involve a number of complex steps. The second method is often referred to as extraction - free and uses physical, enzymatic, or chemical means to lyse cells or microorganisms and amplify nucleic acids without further purification. The second method is simpler and usually faster than the first method, but provides a lower - quality nucleic acid preparation than the first method.

[0003] In a second method, to effectively amplify nucleic acids, amplification inhibitors present in the sample (e.g., PCR inhibitors) must be neutralized so that molecular amplification of the nucleic acids can occur before detection. Further, the nucleic acids must not be degraded so that they can be amplified and detected. For example, metal ions present in cells and / or tissues can inhibit some amplification enzymes and cause nucleic acid degradation. In addition, natural enzymes present in biological samples can either inhibit molecular amplification (e.g., proteases) or degrade nucleic acids (e.g., nucleases). Current means for inactivating these amplification inhibitors, e.g., PCR inhibitors, rely on at least a 10-fold dilution of the sample, which reduces the sensitivity of the overall assay. Further, storage of diluents in fluid cartridges complicates overall manufacturing and reduces the shelf life of point-of-care devices. Thus, a simpler and faster method for effectively treating samples prior to nucleic acid amplification and detection is highly desired. Summary of the Invention Means for Solving the Problems

[0004] Abstract In one aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) heating a processed sample containing nucleic acids in a closed heating chamber under high pressure conditions from a first temperature to a second temperature above 100 degrees Celsius over a ramp time, thereby generating a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample.

[0005] In some embodiments, the processed sample comprises one or more reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0006] In some embodiments, the processed sample comprises a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent.

[0007] In some embodiments, the processing sample comprises a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0008] In some embodiments, the first temperature is from 4°C to 40°C, and the second temperature is from 101°C to 160°C.

[0009] In some embodiments, the gradient time is from 3 to 50 seconds.

[0010] In some embodiments, the step of heating the processing sample is performed at a temperature gradient rate of 5°C per second to 50°C per second.

[0011] In some embodiments, the method further comprises maintaining the processing sample at the second temperature for a holding time of 0 to 120 seconds after step (a) and before step (b) and before the cooling time.

[0012] In some embodiments, the processing sample comprises a body sample containing nucleic acid, where i) the body sample is selected from the group consisting of a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a fecal sample, a cerebrospinal fluid sample, and a urine sample; and ii) the nucleic acid has not been extracted, isolated, or otherwise purified from the body sample.

[0013] In another aspect, the present disclosure provides a method for analyzing nucleic acid, comprising: (a) providing a body sample selected from the group consisting of a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a fecal sample, a cerebrospinal fluid sample, and a urine sample, the body sample containing nucleic acid; (b) heating a processing sample containing the body sample in a closed heating chamber from a first temperature to a second temperature above 100°C over a gradient time under high pressure conditions, thereby generating a heat-treated sample; and (c) analyzing the nucleic acid from the heat-treated sample, wherein the nucleic acid has not been extracted, isolated, or otherwise purified from the body sample.

[0014] In some embodiments, the body sample is a saliva sample or a mucus sample.

[0015] In some embodiments, the bodily sample is collected from a human subject by a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0016] In some embodiments, the bodily sample contains a pathogen or a part thereof.

[0017] In some embodiments, the pathogen or a part thereof is selected from the group consisting of a virus or a part thereof, a bacterium or a part thereof, a protozoan or a part thereof, a yeast or a part thereof, and a fungus or a part thereof.

[0018] In some embodiments, the pathogen is a virus or a part thereof.

[0019] In some embodiments, the virus is SARS-CoV2.

[0020] In some embodiments, the processed sample contains non-lysed cells containing nucleic acids, and the nucleic acids have not been extracted, isolated, or otherwise purified from the heat-treated sample prior to the step of analyzing the nucleic acids from the heat-treated sample.

[0021] In another aspect, the present disclosure provides a method for analyzing nucleic acids, comprising: (a) heating a processed sample containing non-lysed cells containing nucleic acids in a closed heating chamber from a first temperature to a second temperature above 100 degrees Celsius under high pressure conditions to thereby generate a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample, wherein the nucleic acids have not been extracted, isolated, or otherwise purified from the heat-treated sample prior to step (b).

[0022] In some embodiments, the non-lysed cells are selected from the group consisting of bacterial cells, fungal cells, and mammalian cells.

[0023] In some embodiments, the processed sample contains one or more reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0024] In another aspect, the present disclosure provides a method for analyzing a nucleic acid, comprising: (a) heating a processing sample containing the nucleic acid in a closed heating chamber from a first temperature to a second temperature above 100° C. under high pressure conditions to thereby generate a heat-treated sample; and (b) analyzing the nucleic acid from the heat-treated sample, wherein the processing sample contains one or more reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, and a stabilizer.

[0025] In some embodiments, the processing sample contains at least two reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, and a stabilizer.

[0026] In some embodiments, the processing sample contains at least three reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, and a stabilizer.

[0027] In some embodiments, the processing sample contains a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent.

[0028] In some embodiments, the processing sample contains a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, and a stabilizer.

[0029] In some embodiments, the chelating agent has a concentration of 5 wt / vol % to 25 wt / vol % in the processing sample, the single-stranded nucleic acid-binding protein has a concentration of 0.1 μM to 2 μM in the processing sample, the reducing agent has a concentration of 0.1 mM to 2 mM in the processing sample, or the stabilizer has a concentration of 500 ng / mL to 10 mg / mL in the processing sample.

[0030] In some embodiments, the chelating agent is an insoluble chelating agent.

[0031] In some embodiments, the insoluble chelating agent contains a styrene divinylbenzene copolymer.

[0032] In some embodiments, the chelating agent is a soluble chelating agent.

[0033] In some embodiments, the soluble chelating agent contains EDTA.

[0034] In some embodiments, the stabilizer is bovine serum albumin or gelatin.

[0035] In some embodiments, the nucleic acid is DNA.

[0036] In some embodiments, the nucleic acid is RNA.

[0037] In some embodiments, the nucleic acid is selected from the group consisting of viral nucleic acids, bacterial nucleic acids, protozoal nucleic acids, eukaryotic nucleic acids, and fungal nucleic acids.

[0038] In some embodiments, the nucleic acid is a viral nucleic acid.

[0039] In some embodiments, the processed sample contains nucleic acids at 50 copies / mL to 10 9 copies / mL.

[0040] In some embodiments, the processed sample has a volume of 100 μL to 5 mL.

[0041] In some embodiments, the processed sample has a pH of about 8.0 to about 12.0.

[0042] In some embodiments, the processed sample further contains a protease.

[0043] In some embodiments, the processed sample further contains a nuclease inhibitor.

[0044] In some embodiments, the processed sample is heated with a heat source.

[0045] In some embodiments, the heat source includes an induction heater, a heating element, or microwaves.

[0046] In some embodiments, the heat source includes an induction heater.

[0047] In some embodiments, the closed heating chamber includes a thermally conductive material.

[0048] In some embodiments, the closed heating chamber includes a ferromagnetic material.

[0049] In some embodiments, the nucleic acid is not substantially degraded after the heating step.

[0050] In some embodiments, the method further includes amplifying the nucleic acid by polymerase chain reaction (PCR) after the heating step, thereby generating a PCR product.

[0051] In some embodiments, the method further includes detecting the PCR product.

[0052] In some embodiments, the PCR product is detectable after a smaller number of molecular amplification cycles than are required in the absence of heating of the processed sample.

[0053] In some embodiments, the PCR product is detectable after a smaller number of molecular amplification cycles as compared to heating the processed sample at a temperature below 100 degrees Celsius.

[0054] In some embodiments, the PCR product is detectable after the nucleic acid has been amplified using 10 - 55 molecular amplification cycles.

[0055] In some embodiments, the PCR product is detectable after 28 - 35 molecular amplification cycles.

[0056] In some embodiments, the step of analyzing the nucleic acid includes detecting the nucleic acid, sequencing the nucleic acid, or genotyping the nucleic acid.

[0057] In some embodiments, the step of analyzing the nucleic acid comprises detecting the nucleic acid by fluorescence detection.

[0058] In some embodiments, the step of heating the processed sample is performed at a temperature gradient rate of 5 degrees Celsius per second to 20 degrees Celsius per second.

[0059] In another aspect, the present disclosure provides a method for inactivating a molecular amplification inhibitor in a processed sample containing a nucleic acid, the method comprising heating, in a closed heating chamber under high-pressure heating conditions, a processed sample containing i) a nucleic acid and ii) a plurality of molecular amplification inhibitors from a first temperature to a second temperature above 100 degrees Celsius over a gradient time, thereby inactivating the molecular amplification inhibitors among the plurality of amplification inhibitors and generating a heat-treated sample, wherein the nucleic acid is not substantially degraded.

[0060] In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 101 degrees Celsius to 160 degrees Celsius.

[0061] In some embodiments, the method further comprises maintaining the processed sample at the second temperature for a holding time before a cooling time.

[0062] In some embodiments, the holding time is from 0 seconds to 120 seconds.

[0063] In some embodiments, the method inactivates at least 70% of the plurality of molecular amplification inhibitors.

[0064] In some embodiments, the method inactivates at least 90% of the plurality of molecular amplification inhibitors.

[0065] In some embodiments, the processed sample comprises a bodily sample containing a nucleic acid, wherein i) the bodily sample is selected from the group consisting of a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a fecal sample, a cerebrospinal fluid sample, and a urine sample; and ii) the nucleic acid has not been extracted, isolated, or otherwise purified from the bodily sample.

[0066] In some embodiments, the body sample is a saliva sample.

[0067] In some embodiments, the body sample is a mucus sample.

[0068] In some embodiments, the body sample is collected from an animal or human subject.

[0069] In some embodiments, the body sample is collected from an animal or human subject by a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0070] In some embodiments, the processed sample includes non-lysed cells containing nucleic acids.

[0071] In some embodiments, the processed sample includes one or more reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0072] In some embodiments, the processed sample includes at least two reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0073] In some embodiments, the processed sample includes at least three reagents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0074] In some embodiments, the processed sample includes a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent.

[0075] In some embodiments, the processed sample includes a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0076] In some embodiments, the chelating agent has a concentration of 5 wt / vol% to 25 wt / vol% in the processed sample, the single-stranded nucleic acid-binding protein has a concentration of 0.1 μM to 2 μM in the processed sample, the reducing agent has a concentration of 0.1 mM to 2 mM in the processed sample, or the stabilizer has a concentration of 500 ng / mL to 10 mg / mL in the processed sample.

[0077] In some embodiments, the processed sample has a pH of from about 8.0 to about 12.0.

[0078] In some embodiments, the processed sample further comprises a protease.

[0079] In some embodiments, the processed sample further comprises a nuclease inhibitor.

[0080] In some embodiments, the step of heating the processed sample is performed at a temperature gradient rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

[0081] In another aspect, the present disclosure provides a method for analyzing nucleic acids, comprising: (a) heating a processed sample containing nucleic acids in a closed heating chamber under high pressure conditions from a first temperature to a second temperature above 100 degrees Celsius over a gradient time, thereby producing a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample, wherein the heating step is performed at a temperature gradient rate of 5 degrees Celsius per second to 50 degrees Celsius per second. Incorporation by reference

[0082] All publications, patents, and patent applications cited herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0083] The novel features of the present invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which illustrates embodiments that are examples of the principles of the present invention, and the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0095] Detailed Description Provided herein are methods for nucleic acid sample preparation that use temperatures above 100 degrees Celsius to rapidly liberate high-quality nucleic acids from microorganisms or eukaryotic cells in a form that is immediately available for downstream applications. Briefly, a sample collected from a subject (e.g., a body sample) is heated at a temperature above 100 degrees Celsius (e.g., 130 degrees Celsius) for a short period of time (e.g., less than 60 seconds) (e.g., autoclaving) to lyse cells and / or viruses in the sample and liberate the nucleic acids (both DNA and RNA) present therein. The methods described in the present disclosure not only provide an efficient technique for liberating nucleic acids from other components in a biological sample (e.g., proteins or other cellular components), but these methods can also destroy amplification inhibitors present in the biological sample, e.g., PCR inhibitors. Thus, the methods described in the present disclosure provide a nucleic acid sample that can be used for downstream applications (e.g., molecular amplification, nucleic acid detection, nucleic acid sequencing, etc.) without the need for further dilution of the sample. In some cases, the methods described in the present disclosure provide a nucleic acid sample that can be used for molecular amplification and / or PCR analysis without the need to boost the concentration of DNA polymerase, reverse transcriptase, or RNAse inhibitor. Additionally, the methods reach high sensitivity by PCR with thermal cycles of less than 10 minutes when using an ultra-fast thermocycler and are as efficient as more cumbersome nucleic acid extraction methods. Thus, the methods of the present disclosure can save time and resources, e.g., the use of purification kits and expensive enzymes, in the sample preparation step.

[0096] For example, it is known that some nucleic acids, such as RNA, degrade at high temperatures. However, the present disclosure provides the surprising result that nucleic acids (including both DNA and RNA) can be efficiently amplified after being heated to a temperature above 100 degrees Celsius. This finding is particularly surprising with respect to RNA, which has been widely reported to be unstable at high temperatures. In some aspects of the present disclosure, the sample is added to a device or container (e.g., a sealed container) that can sustain a high vapor pressure, and the sample is heated to a temperature above 100 degrees Celsius (e.g., by high-pressure heating) inside the device or container. In some embodiments, prior to the heating step, the container is sealed, thereby preventing steam from escaping during the high-pressure heating step and facilitating an increase in pressure within the container. In some embodiments, the pressure inside the container increases to, for example, about 15 - 40 PSI (including atmospheric pressure). Further, the sealed container prevents the sample solution (e.g., an aqueous sample solution) from boiling, thereby allowing the sample solution to reach a temperature significantly above 100 degrees Celsius (e.g., 130 degrees Celsius). Prevention of boiling of the sample solution can result in cell and / or virus lysis and neutralization of enzyme inhibitors present in the sample without damaging the nucleic acid (DNA or RNA).

[0097] Furthermore, in some embodiments of the present disclosure, the additive is added to the sample (e.g., before, during, or after the heating step). For example, a chelating agent such as Chelex can be added to the sample before high-temperature heating. In some embodiments, the additive is a chelating agent, a reducing agent, a single-stranded nucleic acid-binding protein, an RNase inhibitor, or a combination thereof. In some embodiments, the chelating agent is not added to the sample (e.g., when a reducing agent and / or an RNase inhibitor is added to the sample, the chelating agent can be excluded in sample preparation). The chelating agent can be present during the heating step, or used prior to the heating step and removed before heating, or excluded altogether. Even at the high temperatures used in the methods of the present disclosure, such resins capture metal ions that would otherwise interfere with nucleic acid amplification and / or detection. In some embodiments, the pH is acidic (pH 5). In some other embodiments, the pH is near neutral. In some embodiments, the pH of the sample is basic (e.g., pH 8 or higher), which helps to neutralize RNase activity at the temperatures utilized in the present disclosure. Surprisingly, even under basic conditions and high temperatures, the present disclosure provides a method of sample preparation that does not result in significant degradation of nucleic acids, even in the case of RNA. In some embodiments, a heat-stable single-stranded nucleic acid-binding protein (SSB) is added to the sample mixture before the heating step, with or without a chelating agent, such that the pH is neutralized to the pH at which the nuclease enzyme is active, and further protects the nucleic acid (e.g., RNA) during the heating step.

[0098] RNase can sometimes survive severe heating conditions. Therefore, in some embodiments, a reducing agent, such as an additive like DTT or TCEP, is added, with or without a chelating agent, to completely neutralize RNase using the methods of the present disclosure.

[0099] The present disclosure provides a sample preparation method that provides high-quality nucleic acid sample preparation, and the nucleic acid sample preparation from this method can be readily used for downstream applications such as molecular amplification and detection. The overall process can use a shorter workflow in a shorter time compared to conventional sample processing methods, providing a more efficient method for nucleic acid sample preparation.

[0100] Definitions Unless otherwise defined, all technical and scientific terms or terminology used herein, including all specialized terms, notations, and other technical and scientific terms or glossaries, are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

[0101] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, a range description is to be considered as specifically disclosing not only all the possible sub-ranges, but also each individual numerical value within that range. For example, a range description such as 1 - 6 is to be considered as specifically disclosing sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as each individual number within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0102] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples including mixtures thereof.

[0103] The terms "determining," "measuring," "assessing," "evaluating," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present or not (e.g., detecting). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. Evaluating can be relative or absolute. "Detecting the presence of" can, depending on the context, include determining the amount of what is present in addition to determining whether it is present or not.

[0104] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" can be a biological entity that contains genetic material. The biological entity can be a plant, an animal, or a microorganism including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, a cell, or a fragment thereof that is derived from a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal, such as a human. A subject can be diagnosed or suspected of having a high risk of a disease. In some cases, a subject may not necessarily be diagnosed or suspected of having a high risk of a disease.

[0105] As used herein, the term "about" in reference to a number refers to that number plus or minus 10% of that number. The term "about" in reference to a range refers to the range that is 10% minus of the lowest value and 10% plus of the highest value thereof.

[0106] As used herein, the term "molecular amplification" refers to an assay, method, or test used to detect nucleic acids in a sample. This can be used, for example, in experimental research, clinical drug development, infectious disease diagnosis, gene cloning, and industrial quality control. Molecular amplification can also be used as part of a diagnostic test. The term "molecular amplification" as referred to herein is intended to encompass all methods designed to amplify (e.g., replicate, duplicate, etc.) nucleic acids, thereby generating more copies of the nucleic acids in the sample. Molecular amplification includes nucleic acid amplification, enzymatic amplification (e.g., PCR), isothermal amplification, and / or other alternative amplification methods developed in this field.

[0107] As used herein, the term "polymerase chain reaction" or "PCR" refers to a type of molecular or nucleic acid amplification that amplifies or generates more copies of a nucleic acid template. The method of PCR can be used in conjunction with methods for detecting, identifying, and / or quantifying nucleic acids. The term "PCR" as used herein is intended to encompass all different types of PCR, including, for example, sequential PCR and real-time PCR ("RT-PCR").

[0108] As used herein, the term "biological sample" means a sample containing nucleic acids / biological agents, such as clinical samples (e.g., cell fractions, mucosa, nasal swabs, whole blood, plasma, serum, urine, tissue, cells, etc.), agricultural samples, environmental samples (e.g., soil, mud, minerals, water, air), food samples, forensic samples, or any other biological sample. The sample may contain, for example, infectious agents such as viral, bacterial, or parasitic infection pathogens. "Whole blood" means blood containing white blood cells and red blood cells, platelets, plasma, and any infectious agents that may be present, such as blood collected by venipuncture. Clinical samples may be of human or animal origin. The sample to be analyzed can be natural, solid, or liquid. When solid materials are used, it is understood that they are first dissolved in a suitable solution as known in the art.

[0109] As used herein, the term "chelating agent" refers to an agent used to remove metal ions in a solution. Chelating agents include, for example, soluble chelating agents (e.g., agents capable of forming water-soluble complexes with metal ions) such as sodium tripolyphosphate, EDTA, DTPA, NTA, citrate, etc. Chelating agents further include, for example, insoluble chelating agents (e.g., agents capable of forming insoluble complexes with metal ions) such as trisodium phosphate, zeolite A, etc. In some embodiments, the chelating agent is used to remove metal ion impurities in a sample or to inactivate and / or inhibit a molecular amplification inhibitor, resulting in higher efficiency in the molecular amplification of the sample. The quality of a nucleic acid sample can be increased using a chelating agent prior to performing molecular amplification.

[0110] As used herein, the term "reducing agent" refers to an agent that donates electrons to an electron acceptor. In some embodiments, the term "reducing agent" refers to an agent used to reduce the disulfide bonds in a protein. Reducing agents include, among others, for example, 2-mercaptoethanol, 2-mercaptoethylamine-HCl, TCEP, cysteine-HCl, dithiothreitol (DTT), TCEP-HCl, thiol-based reducing agents, guanidine-HCl, and urea. Other reducing agents known in the art can be used as described in this disclosure.

[0111] As used herein, the term "high pressure" or "high-pressure" refers to a state where the pressure is greater or higher than the normal pressure at the same elevation level.

[0112] As used herein, the term "nucleic acid" refers to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA-RNA hybrids, and analogs of DNA or RNA generated using nucleotide analogs. Nucleic acid molecules can include nucleotides, oligonucleotides, double-stranded DNA, single-stranded DNA, multi-stranded DNA, complementary DNA, genomic DNA, non-coding DNA, messenger RNA (mRNA), single-stranded RNA, microRNA (miRNA), and small nuclear RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), heterogeneous nuclear RNA (hnRNA), or small hairpin type (shRNA).

[0113] Method for sample preparation The present disclosure provides a method for sample preparation for application to a biological sample containing nucleic acid. Examples of biological samples containing nucleic acid can be body samples (e.g., saliva or mucus) or cells (e.g., bacterial cells, fungal cells, or mammalian cells). The present disclosure provides a method for sample preparation of nucleic acid in a body sample that does not include extraction, isolation, or other forms of purification of nucleic acid from the body sample. The present disclosure provides a method for inactivating molecular amplification inhibitors (e.g., RNAse present in saliva or mucus) in a sample, thereby preparing a sample for further processing.

[0114] The present disclosure further provides a method for analyzing nucleic acid in a biological sample, including the step of preparing a sample using the method of the present disclosure and then analyzing the nucleic acid. The sample preparation method described herein can prepare a sample for molecular amplification of nucleic acid in the sample. In some embodiments, the method described herein can improve the efficiency of nucleic acid analysis by improving the efficiency of molecular amplification. The method described herein can reduce the degradation degree of nucleic acid and improve the detectability of nucleic acid or its molecular amplification product for analysis.

[0115] The step of analyzing the nucleic acid can be performed using any of several techniques known in the art (e.g., sequencing the nucleic acid (e.g., sequencing by synthesis, sequencing by hybridization, nanopore sequencing, etc.), genotyping the amino acid (e.g., genotyping by hybridization, genotyping by sequencing, etc.), or detecting the nucleic acid (e.g., can be performed using detection by hybridization, antibody binding, fluorescence, radioisotope detection, etc.). For example, in some embodiments, detecting the nucleic acid includes hybridizing the nucleic acid to a fluorescently labeled nucleic acid comprising a sequence that is complementary to at least a portion of the nucleic acid using methods known in the art.

[0116] In some embodiments, the step of analyzing the nucleic acid includes one or more of: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the step of analyzing the nucleic acid includes detecting the nucleic acid. In some embodiments, the step of analyzing the nucleic acid includes sequencing the nucleic acid. In some embodiments, the step of analyzing the nucleic acid includes genotyping the nucleic acid.

[0117] In some embodiments, the step of analyzing the nucleic acid includes analyzing the molecular amplification product of the nucleic acid (e.g., the DNA copy of RNA produced during PCR amplification of RNA in a sample using reverse transcriptase). For example, in some embodiments, detecting the nucleic acid includes detecting the molecular amplification product of the nucleic acid. In some embodiments, sequencing the nucleic acid includes sequencing the molecular amplification product of the nucleic acid. In some embodiments, genotyping the nucleic acid includes genotyping the molecular amplification product of the nucleic acid. Those skilled in the art will understand that many means for analyzing nucleic acids are known in the art and all of them are compatible with the methods of the present disclosure and are contemplated herein.

[0118] In some embodiments, molecular amplification includes enzymatic amplification. In some embodiments, molecular amplification includes isothermal amplification. In some embodiments, nucleic acid amplification includes polymerase chain reaction ("PCR"), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling cycle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HAD), ramification amplification method (RAM), transcription-mediated assay (TMA), nicking enzyme amplification reaction (NEAR), recombinase polymerase amplification (RPA), or whole genome amplification (WGA).

[0119] In some embodiments, molecular amplification includes polymerase chain reaction ("PCR"). In some embodiments, PCR includes reverse transcriptase-polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), quantitative real-time PCR (qPCR), digital PCR (dPCR), digital droplet PCR (ddPCR), microfluidic PCR, multiplex PCR, variable number tandem repeat (VNTR) PCR, asymmetric PCR, nested PCR, quantitative PCR, hot start PCR, touchdown PCR, assembly PCR, colony PCR, suicide PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), rapid amplification of cDNA ends (RACE) PCR, two-sided PCR, ligation-mediated PCR, methylation-specific PCR (MSP), InterSequence-Specific PCR (or ISSR-PCR), RNase H-dependent PCR (rhPCR), or vectorette PCR.

[0120] In some embodiments, nucleic acids can be analyzed after molecular amplification of the nucleic acids (e.g., PCR). In some embodiments, nucleic acids are analyzed as part of a downstream application. In some embodiments, the downstream application includes molecular amplification or sequencing. In some embodiments, the downstream application includes probe hybridization and / or detecting a probe.

[0121] In one aspect, the method of the present disclosure includes a step of heating a processed sample. As used herein, a "processed sample" is a sample (e.g., an aqueous solution or suspension) containing at least nucleic acids and, optionally, reagents such as enzymes or chelating agents. In certain embodiments, the processed sample may be a biological sample of nucleic acids collected from a subject, diluted with water or a buffer, and containing one or more reagents as needed. The biological sample may be a bodily sample, such as saliva or mucus. The biological sample may contain cells (e.g., non-lysed cells). In certain embodiments, a sample of nucleic acids collected from a subject is isolated or purified before dilution and / or addition of reagents, e.g., isolated or purified from cells, proteins, or other biological agents in the biological sample. In certain embodiments, a sample of nucleic acids collected from a subject is neither isolated nor purified before dilution and / or addition of reagents, e.g., not isolated or purified from cells, proteins, or other biological agents in the biological sample.

[0122] In some embodiments, the method includes heating a processing sample in a closed heating chamber to a temperature above 100° C. under high-pressure heating conditions. For example, the closed heating chamber may substantially prevent air and vapor from entering or exiting the chamber. In some cases, there may be a negligible air flow in and out of the closed heating chamber. The closed heating chamber may remain closed during high-pressure heating. In some embodiments, the "high-pressure heating conditions" referred to herein are conditions under which heating generates a higher pressure inside the closed heating chamber than outside the closed heating chamber. In some embodiments, the temperature referred to herein is the average temperature during the step of heating the sample or a part thereof. In some embodiments, the temperature referred to herein is the temperature inside the closed container. In some embodiments, the temperature referred to herein is the temperature of the heating device used to heat the sample under high pressure. In some cases, the temperature is estimated using an internal temperature sensor inside the closed container. In some cases, the internal temperature is correlated with an external temperature infrared sensor that evaluates the external temperature of the closed container. In some embodiments, the heating of the processing sample is performed at a temperature gradient rate, for example, from 6° C. per second to 20° C. per second. In some embodiments, the method of the present disclosure includes heating a processing sample from a first temperature to a second temperature above 100° C. over a gradient time.

[0123] In some embodiments, the method of the present disclosure includes analyzing nucleic acids in a body sample selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In one embodiment, the method includes providing a body sample and heating a processing sample containing the body sample. In some embodiments, the nucleic acids have not been extracted, isolated, or otherwise purified from the body sample.

[0124] In some embodiments, the method of the present disclosure includes heating a processed sample containing non-lysed cells containing nucleic acids, thereby generating a heat-treated sample. In some embodiments, the method further includes analyzing the nucleic acids. In some embodiments, the nucleic acids are not extracted, isolated, or otherwise purified from the heat-treated sample prior to the step of analyzing the nucleic acids.

[0125] In some embodiments, the processed sample containing nucleic acids further includes one or more reagents selected from chelating agents, single-stranded nucleic acid-binding proteins, and reducing agents. In some embodiments, the processed sample is a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent.

[0126] In some embodiments, the present disclosure provides a method of inactivating a molecular amplification inhibitor in a processed sample containing nucleic acids. In some embodiments, the method includes heating a processed sample containing (i) nucleic acids and (ii) a plurality of molecular amplification inhibitors. In one embodiment, the method includes heating the processed sample in a closed heating chamber under high-pressure heating conditions to a temperature above 100° C., thereby inactivating the molecular amplification inhibitor among the plurality of amplification inhibitors and generating a heat-treated sample. In another embodiment, the method includes heating the processed sample in a closed heating chamber under high-pressure heating conditions from a first temperature to a second temperature above 100° C. over a gradient time, thereby inactivating the molecular amplification inhibitor among the plurality of amplification inhibitors and generating a heat-treated sample, wherein the nucleic acids are not substantially degraded. In some embodiments, the method further includes detecting the nucleic acids.

[0127] In one aspect, the method described herein includes heating a treatment sample in a closed heating chamber to a temperature above 100 degrees Celsius under high pressure conditions. In some embodiments, the method includes heating the treatment sample under high pressure to a temperature above 100 degrees Celsius. The temperature may be above the boiling point of water. In some embodiments, the treatment sample does not boil at a temperature above 100 degrees Celsius under high pressure conditions. In some embodiments, the method includes heating the treatment sample under high pressure to a temperature between 100 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 101 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 105 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 110 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 120 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 130 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 140 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 150 degrees Celsius and 160 degrees Celsius. In some embodiments, the temperature is between 100 degrees Celsius and 140 degrees Celsius. In some embodiments, the temperature is between 110 degrees Celsius and 140 degrees Celsius. In some embodiments, the temperature is between 120 degrees Celsius and 140 degrees Celsius. In some embodiments, the temperature is between 130 degrees Celsius and 140 degrees Celsius. In some embodiments, the temperature is about 110 degrees Celsius. In some embodiments, the temperature is about 120 degrees Celsius. In some embodiments, the temperature is about 130 degrees Celsius. In some embodiments, the temperature is about 140 degrees Celsius. In some embodiments, the temperature is about 150 degrees Celsius. In some embodiments, the temperature is about 160 degrees Celsius.

[0128] In some embodiments, heating of the processing sample occurs during a first period. In some embodiments, the first period is, for example, about 10 seconds, about 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 120 seconds, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 180 seconds, 190 seconds, 200 seconds or 300 seconds. In some embodiments, the first period is, for example, 1 second to 300 seconds, 5 seconds to 300 seconds, 30 seconds to 300 seconds, 40 seconds to 300 seconds, 50 seconds to 300 seconds, 60 seconds to 300 seconds, 70 seconds to 300 seconds, 80 seconds to 300 seconds, 90 seconds to 300 seconds, 100 seconds to 300 seconds, 110 seconds to 300 seconds, 120 seconds to 300 seconds, or 200 seconds to 300 seconds. In some embodiments, the first period is, for example, 10 seconds to 180 seconds, 20 seconds to 180 seconds, 30 seconds to 180 seconds, 40 seconds to 180 seconds, 50 seconds to 180 seconds, 60 seconds to 180 seconds, 70 seconds to 180 seconds, 80 seconds to 180 seconds, 90 seconds to 180 seconds, 100 seconds to 180 seconds, 110 seconds to 180 seconds, or 120 seconds to 180 seconds. In some embodiments, the first period is, for example, 10 seconds to 120 seconds, 20 seconds to 120 seconds, 30 seconds to 120 seconds, 40 seconds to 120 seconds, 50 seconds to 120 seconds, 60 seconds to 120 seconds, 70 seconds to 120 seconds, 80 seconds to 120 seconds, 90 seconds to 120 seconds, 100 seconds to 120 seconds, or 110 seconds to 120 seconds. In some embodiments, the first period is, for example, 30 seconds to 90 seconds, 40 seconds to 90 seconds, 50 seconds to 90 seconds, 60 seconds to 90 seconds, 70 seconds to 90 seconds, or 80 seconds to 90 seconds. In some embodiments, the first period is, for example, 10 seconds to 60 seconds, 20 seconds to 60 seconds, 30 seconds to 60 seconds, 40 seconds to 60 seconds, or 50 seconds to 60 seconds.

[0129] In some embodiments, the first period is, for example, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes. In some embodiments, the first period is, for example, 1 minute to 2 minutes, 1 minute to 3 minutes, 1 minute to 4 minutes, 1 minute to 5 minutes, 1 minute to 6 minutes, 1 minute to 7 minutes, 1 minute to 8 minutes, 1 minute to 9 minutes, or 1 minute to 10 minutes. In some embodiments, the first period is, for example, 1 minute to 5 minutes, 2 minutes to 5 minutes, 3 minutes to 5 minutes, 4 minutes to 5 minutes.

[0130] In some embodiments, the method further includes maintaining the processed sample at that temperature for a holding time after heating the processed sample to a temperature above 100 degrees Celsius and before a cooling time. In some embodiments, the holding time is from 0 seconds to 300 seconds, from 0 seconds to 250 seconds, from 0 seconds to 200 seconds, from 0 seconds to 150 seconds, from 0 seconds to 120 seconds, from 0 seconds to 100 seconds, from 0 seconds to 50 seconds, from 0 seconds to 40 seconds, from 0 seconds to 30 seconds, from 0 seconds to 20 seconds, or from 1 to 10 seconds. In some embodiments, the holding time is from 5 seconds to 300 seconds, from 5 seconds to 250 seconds, from 5 seconds to 200 seconds, from 5 seconds to 150 seconds, from 5 seconds to 100 seconds, from 5 seconds to 50 seconds, from 5 seconds to 40 seconds, from 5 seconds to 30 seconds, from 5 seconds to 20 seconds, or from 5 to 10 seconds.

[0131] During the holding time, the closed heating chamber may remain closed (e.g., substantially preventing air and vapor from entering or exiting the chamber).

[0132] In some embodiments, the cooling time is from 5 seconds to 300 seconds. In some embodiments, the cooling time is from 30 seconds to 300 seconds. In some embodiments, the cooling time is from 60 seconds to 300 seconds. In some embodiments, the cooling time is from 90 seconds to 300 seconds. In some embodiments, the cooling time is from 120 seconds to 300 seconds. In some embodiments, the cooling time is from 150 seconds to 300 seconds. In some embodiments, the cooling time is from 180 seconds to 300 seconds. In some embodiments, the cooling time is from 240 seconds to 300 seconds. In some embodiments, the cooling time is from 270 seconds to 300 seconds.

[0133] In some embodiments, the cooling time is from 10 seconds to 120 seconds. In some embodiments, the cooling time is from 30 seconds to 120 seconds. In some embodiments, the cooling time is from 60 seconds to 120 seconds. In some embodiments, the cooling time is from 90 seconds to 120 seconds. During the cooling time, the closed heating chamber may remain closed (e.g., substantially preventing air and vapor from entering or exiting the chamber). In some embodiments, the nucleic acid is not substantially degraded after the heating step.

[0134] In some embodiments, the step of heating the processed sample containing the nucleic acid is performed at a temperature gradient rate. In some embodiments, the temperature gradient rate is calculated as the time derivative of the temperature. In some embodiments, the temperature gradient rate is calculated as the average rate of change of the temperature with respect to time. In some embodiments, the temperature gradient rate is at least 0.5 degrees Celsius per second, at least 1 degree Celsius per second, at least 2 degrees Celsius per second, at least 3 degrees Celsius per second, at least 4 degrees Celsius per second, at least 5 degrees Celsius per second, at least 6 degrees Celsius per second, at least 7 degrees Celsius per second, at least 8 degrees Celsius per second, at least 9 degrees Celsius per second, at least 10 degrees Celsius per second, at least 11 degrees Celsius per second, at least 12 degrees Celsius per second, at least 13 degrees Celsius per second, at least 14 degrees Celsius per second, at least 15 degrees Celsius per second, at least 16 degrees Celsius per second, at least 17 degrees Celsius per second, at least 18 degrees Celsius per second, at least 19 degrees Celsius per second, at least 20 degrees Celsius per second, at least 25 degrees Celsius per second, at least 27.5 degrees Celsius per second, at least 30 degrees Celsius per second, at least 32.5 degrees Celsius per second, at least 35 degrees Celsius per second, at least 37.5 degrees Celsius per second, at least 40 degrees Celsius per second, at least 42.5 degrees Celsius per second, at least 45 degrees Celsius per second, at least 47.5 degrees Celsius per second, or at least 50 degrees Celsius per second.

[0135] In some embodiments, the temperature gradient rate is from 0.5 degrees Celsius per second to 50 degrees Celsius per second, from 0.5 degrees Celsius per second to 40 degrees Celsius per second, from 0.5 degrees Celsius per second to 35 degrees Celsius per second, from 0.5 degrees Celsius per second to 30 degrees Celsius per second, from 0.5 degrees Celsius per second to 25 degrees Celsius per second, from 0.5 degrees Celsius per second to 20 degrees Celsius per second, or from 0.5 degrees Celsius per second to 15 degrees Celsius per second.

[0136] In some embodiments, the rate of temperature gradient is from 2 degrees Celsius per second to 50 degrees Celsius per second, from 2 degrees Celsius per second to 40 degrees Celsius per second, from 2 degrees Celsius per second to 35 degrees Celsius per second, from 2 degrees Celsius per second to 30 degrees Celsius per second, from 2 degrees Celsius per second to 25 degrees Celsius per second, from 2 degrees Celsius per second to 20 degrees Celsius per second, or from 2 degrees Celsius per second to 15 degrees Celsius per second.

[0137] In some embodiments, the rate of temperature gradient is from 5 degrees Celsius per second to 50 degrees Celsius per second, from 5 degrees Celsius per second to 40 degrees Celsius per second, from 5 degrees Celsius per second to 35 degrees Celsius per second, from 5 degrees Celsius per second to 30 degrees Celsius per second, from 5 degrees Celsius per second to 25 degrees Celsius per second, from 5 degrees Celsius per second to 20 degrees Celsius per second, or from 5 degrees Celsius per second to 15 degrees Celsius per second. In some embodiments, the nucleic acid is not substantially degraded after the heating step.

[0138] In one aspect, the method described herein includes heating a processing sample containing the nucleic acid in a closed heating chamber under high pressure conditions from a first temperature to a second temperature above 100 degrees Celsius over a gradient time, thereby producing a heat-treated sample. The closed heating chamber may remain closed during heating from the first temperature to the second temperature (e.g., substantially preventing air from entering or exiting the chamber). In some embodiments, the processing sample does not boil at the second temperature above 100 degrees Celsius under high pressure conditions. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 100 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 101 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 105 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 110 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 120 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 130 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is from 4 degrees Celsius to 40 degrees Celsius, and the second temperature is from 140 degrees Celsius to 160 degrees Celsius.

[0139] In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 100°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 101°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 105°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 110°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 120°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 130°C to 160°C. In some embodiments, the first temperature is from 20°C to 30°C, and the second temperature is from 140°C to 160°C.

[0140] In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 100 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 101 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 105 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 110 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 120 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 130 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 140 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 150 degrees Celsius. In some embodiments, the first temperature ranges from 4 degrees Celsius to 40 degrees Celsius, and the second temperature and the second temperature are at least 160 degrees Celsius.

[0141] In some embodiments, the gradient time is 3 to 100 seconds, 6 to 100 seconds, 7 to 100 seconds, 8 to 100 seconds, 9 to 100 seconds, or 10 to 100 seconds. In some embodiments, the gradient time is 3 to 50 seconds, 6 to 50 seconds, 7 to 50 seconds, 8 to 50 seconds, 9 to 50 seconds, or 10 to 50 seconds.

[0142] In some embodiments, the method of analyzing a nucleic acid further includes maintaining the processed sample at a second temperature during a holding time after (a) and before the cooling time. In some embodiments, the holding time is from 0 seconds to 300 seconds. In some embodiments, the holding time is from 5 seconds to 300 seconds. In some embodiments, the holding time is from 30 seconds to 300 seconds. In some embodiments, the holding time is from 60 seconds to 300 seconds. In some embodiments, the holding time is from 90 seconds to 300 seconds. In some embodiments, the holding time is from 120 seconds to 300 seconds. In some embodiments, the holding time is from 150 seconds to 300 seconds. In some embodiments, the holding time is from 180 seconds to 300 seconds. In some embodiments, the holding time is from 240 seconds to 300 seconds. In some embodiments, the holding time is from 270 seconds to 300 seconds.

[0143] In some embodiments, the holding time is from 0 seconds to 120 seconds. In some embodiments, the holding time is from 10 seconds to 120 seconds. In some embodiments, the holding time is from 30 seconds to 120 seconds. In some embodiments, the holding time is from 60 seconds to 120 seconds. In some embodiments, the holding time is from 90 seconds to 120 seconds. During the holding time, the closed heating chamber may remain closed (e.g., substantially preventing air and vapor from entering or exiting the chamber).

[0144] In some embodiments, the cooling time is from 0 seconds to 300 seconds. In some embodiments, the cooling time is from 5 seconds to 300 seconds. In some embodiments, the cooling time is from 30 seconds to 300 seconds. In some embodiments, the cooling time is from 60 seconds to 300 seconds. In some embodiments, the cooling time is from 90 seconds to 300 seconds. In some embodiments, the cooling time is from 120 seconds to 300 seconds. In some embodiments, the cooling time is from 150 seconds to 300 seconds. In some embodiments, the cooling time is from 180 seconds to 300 seconds. In some embodiments, the cooling time is from 240 seconds to 300 seconds. In some embodiments, the cooling time is from 270 seconds to 300 seconds.

[0145] In some embodiments, the cooling time is from 10 seconds to 120 seconds. In some embodiments, the cooling time is from 30 seconds to 120 seconds. In some embodiments, the cooling time is from 60 seconds to 120 seconds. In some embodiments, the cooling time is from 90 seconds to 120 seconds. During the cooling time, the closed heating chamber may remain closed (e.g., substantially preventing air and vapor from entering or exiting the chamber).

[0146] In some aspects, the step of heating the sample to be processed in the closed chamber generates pressure inside the chamber. In some embodiments, the pressure inside the chamber is, for example, 1 - 200 PSI, 10 - 200 PSI, 20 - 200 PSI, 30 - 200 PSI, 40 - 200 PSI, 50 - 200 PSI, 10 - 100 PSI, 20 - 100 PSI, 30 - 100 PSI, 40 - 100 PSI, 50 - 100 PSI, 60 - 100 PSI, 70 - 100 PSI, 80 - 100 PSI, or 90 - 100 PSI, each of which is relative to 1 atm. In some embodiments, the pressure inside the chamber is, for example, 10 - 100 PSI, 10 - 90 PSI, 10 - 80 PSI, 10 - 70 PSI, 10 - 60 PSI, 10 - 50 PSI, 10 - 40 PSI, 10 - 30 PSI, 10 - 20 PSI, 20 - 100 PSI, 20 - 90 PSI, 20 - 80 PSI, 20 - 70 PSI, 20 - 60 PSI, 20 - 50 PSI, 20 - 40 PSI, 20 - 30 PSI, 30 - 100 PSI, 30 - 90 PSI, 30 - 80 PSI, 30 - 70 PSI, 30 - 60 PSI, 30 - 50 PSI, or 30 - 40 PSI, each of which is relative to 1 atm. In some embodiments, the pressure inside the chamber is 40 - 60 PSI relative to 1 atm. In some embodiments, the pressure inside the chamber is 40 - 50 PSI relative to 1 atm. In some embodiments, the nucleic acid is not substantially degraded after the heating step.

[0147] Although not limited thereto, any means for heating a sample can be used, including contact heating, induction heating, microwave heating, nanofotonic heating, or any other heating means known in the art in a dry heat block. In some embodiments, the sample to be processed is heated with a heat source. In some embodiments, the heat source includes an induction heater, a heating element, or microwaves. For example, in some embodiments, a heating block or heating bath can be used by filling it, for example, with pre-heated aluminum beads. In some embodiments, the heating of the biological sample is high-pressure heating. In some embodiments, the sample to be processed is heated under high pressure with a heat source.

[0148] For example, in some embodiments, the sample to be processed is heated using a heating block or heating bath that is pre-heated to a temperature in the range of 100°C to 300°C, or more preferably 100°C to 150°C, where a high-pressure heating container is installed and left for 1 second to 10 minutes, for example, after reaching 100°C. In another embodiment, the container is heated under high pressure for 20 to 40 seconds after reaching a temperature of 100°C or higher.

[0149] In another embodiment of the present disclosure, heat can be applied to the sample by induction heating. In some embodiments, the container can be made of a paramagnetic material or a paramagnetic material can be placed in direct contact with the sample inside a heating container made of a ferromagnetic heat-resistant material such as a plastic polymer. This provides an extreme temperature gradient rate that allows the sample to exceed 100°C in less than 30 seconds, which enables complete sample preparation in 1 minute or less.

[0150] In some embodiments, a laser can be used to heat a processing sample inside a container at a temperature above 100 degrees Celsius (e.g., high-pressure heating). Nanoparticles can be added to the heating system to capture laser energy and directly transfer it to the sample. This technique provides a very high heating gradient rate that is compatible with superheated sample preparation. In some embodiments, the heat source is a laser. In some embodiments, nanoparticles are added to the laser.

[0151] In some aspects, the present disclosure provides a method of heating a processing sample comprising a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is derived from a eukaryotic or prokaryotic cell. In some embodiments, the nucleic acid is derived from a virus. In some embodiments, the nucleic acid is selected from viral nucleic acid, bacterial nucleic acid, protozoal nucleic acid, eukaryotic nucleic acid, and fungal nucleic acid. In some embodiments, the nucleic acid is viral nucleic acid.

[0152] In some embodiments, a processing sample comprising a bodily sample containing a nucleic acid is heated under high-pressure conditions in a closed heating chamber. In some embodiments, the bodily sample comprises a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymph, bile, synovial fluid, cyst fluid, ascites, pleural effusion, ocular fluid, interstitial fluid, cervical mucus, and urine. In some embodiments, the bodily sample is selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In some embodiments, the bodily sample comprises mucus. In some embodiments, the bodily sample comprises a bodily fluid sample, tissue, or cell of a subject. In some embodiments, the nucleic acid has not been extracted, isolated, or otherwise purified from the bodily sample.

[0153] In some embodiments, the bodily sample may be collected from a subject (e.g., a human). The bodily sample may be collected from the subject by a nasopharyngeal swab, a cervical swab, or a nasal swab. In some cases, the bodily sample may contain a pathogen or a part thereof. In some embodiments, the pathogen or a part thereof is selected from a virus or a part thereof, a bacterium or a part thereof, a protozoan or a part thereof, a yeast or a part thereof, and a fungus or a part thereof. In some embodiments, the pathogen is a blood-bone pathogen or a part thereof. In some embodiments, the pathogen is a respiratory pathogen or a part thereof.

[0154] In some embodiments, a processed sample containing a bodily sample comprising nucleic acid is heated in a closed heating chamber under high pressure conditions to a temperature above 100 degrees Celsius, thereby generating a heat-treated sample. In some embodiments, the nucleic acid is analyzed after high-pressure heating. In some cases, the nucleic acid has not been extracted, isolated, or otherwise purified from the bodily sample. For example, in some cases, the nucleic acid has not been extracted by phenol-chloroform extraction, purified by a commercially available nucleic acid purification kit, or purified by column chromatography.

[0155] In some embodiments, a processed sample containing cells containing nucleic acids is heated under high pressure conditions in a closed heating chamber. The cells may be non-lysed cells. In some embodiments, the cells are embedded in a biological matrix, such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine, or saliva. In some embodiments, the cells are embedded in nasal mucus. In some embodiments, the cells are bacterial cells, such as B. subtilis, E. coli, or S. pyogenes. In some embodiments, the cells are fungal cells, such as C. albicans. In some embodiments, the cells are yeast cells, such as S. cerevisiae. In some embodiments, the cells are mammalian cells, such as Chinese hamster ovary cells, BHK cells, or mouse C127 cells. In some embodiments, the cells are human cells, such as HeLa cells. In some embodiments, the processed sample contains bacterial spores, such as B. cereus bacterial spores.

[0156] In some embodiments, a processed sample containing non-lysed cells containing nucleic acids is heated in a closed heating chamber to a temperature above 100 degrees Celsius under high pressure conditions, thereby producing a heat-treated sample. In some embodiments, the nucleic acids are analyzed after high-pressure heating. In some cases, the nucleic acids have not been extracted, isolated, or otherwise purified from the heat-treated sample prior to the step of analyzing the nucleic acids.

[0157] In some aspects, the present disclosure provides a method for inactivating a molecular amplification inhibitor in a processed sample containing nucleic acids. In some cases, the molecular amplification inhibitor may be an agent that binds to nucleic acids. The molecular amplification inhibitor may be an agent that degrades nucleic acids. In some cases, the molecular amplification inhibitor may be a nuclease. In some cases, the molecular amplification inhibitor is DNase. In other cases, the molecular amplification inhibitor is RNase.

[0158] In some embodiments, the method comprises heating a processed sample comprising i) a nucleic acid and ii) a plurality of molecular amplification inhibitors. In one embodiment, the method comprises heating the processed sample in a closed heating chamber to a temperature above 100° C. under high-pressure heating conditions, thereby inactivating the molecular amplification inhibitors among the plurality of amplification inhibitors and generating a heat-treated sample. In another embodiment, the method comprises heating the processed sample in a closed heating chamber from a first temperature to a second temperature above 100° C. over a gradient time under high-pressure heating conditions, thereby inactivating the molecular amplification inhibitors among the plurality of amplification inhibitors and generating a heat-treated sample, wherein the nucleic acid is not substantially degraded. In some embodiments, the nucleic acid is degraded by, for example, 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the method further comprises amplifying the nucleic acid using molecular amplification. In some embodiments, the method further comprises detecting the nucleic acid.

[0159] In some embodiments, the method inactivates at least one molecular amplification inhibitor in the processed sample. In some embodiments, the method inactivates at least 60% of the plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 70% of the plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 80% of the plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 90% of the plurality of molecular amplification inhibitors.

[0160] In some embodiments, the nucleic acid is not substantially degraded after heating, such as by high-pressure heating. In some embodiments, after high-pressure heating, for example, 20% or less, 15% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the nucleic acid is degraded. In some embodiments, after high-pressure heating, for example, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 99.5% of the nucleic acid remains intact.

[0161] In some embodiments, the nucleic acid is analyzed after high-pressure heating. In some embodiments, the method of analyzing the nucleic acid comprises, after high-pressure heating, amplifying the nucleic acid by molecular amplification (e.g., by polymerase) using any of the methods disclosed elsewhere herein. Molecular amplification may be performed during real-time polymerase chain reaction (real-time PCR), transcription-mediated amplification (TMA), or loop-mediated isothermal amplification (LAMP). Molecular amplification of the nucleic acid may produce a molecular amplification product. In some embodiments, the method of analyzing the nucleic acid further comprises, after the step of heating, amplifying the nucleic acid by polymerase chain reaction (PCR) to thereby produce a PCR product. In some embodiments, the method further comprises detecting the PCR product. In some cases, the PCR product is detected by a fluorescence signal emitted during amplification.

[0162] In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after a smaller number of molecular amplification cycles than would be required in the absence of high temperature heating of the processed sample. In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after a smaller number of molecular amplification cycles as compared to heating the processed sample at a temperature below 100 degrees Celsius. In some embodiments, the nucleic acid is detectable after a smaller number of molecular amplification cycles as compared to heating the sample at the same time at a temperature below the boiling point of the sample.

[0163] In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after 10 to 50 molecular amplification cycles, 15 to 50 molecular amplification cycles, 20 to 50 molecular amplification cycles, 25 to 50 molecular amplification cycles, 26 to 50 molecular amplification cycles, 27 to 50 molecular amplification cycles, 28 to 50 molecular amplification cycles, 29 to 50 molecular amplification cycles, or 30 to 50 molecular amplification cycles.

[0164] In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after 10 to 40 molecular amplification cycles, 20 to 40 molecular amplification cycles, 25 to 40 molecular amplification cycles, 26 to 40 molecular amplification cycles, 27 to 40 molecular amplification cycles, 28 to 40 molecular amplification cycles, 29 to 40 molecular amplification cycles, or 30 to 40 molecular amplification cycles.

[0165] In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after 10 - 35 molecular amplification cycles, 20 - 35 molecular amplification cycles, 25 - 35 molecular amplification cycles, 26 - 35 molecular amplification cycles, 27 - 35 molecular amplification cycles, 28 - 35 molecular amplification cycles, 29 - 35 molecular amplification cycles, or 30 - 35 molecular amplification cycles. In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after 28 - 35 molecular amplification cycles. In some embodiments, the nucleic acid is detectable, for example, after 25 - 35 molecular amplification cycles, 25 - 34 molecular amplification cycles, 25 - 33 molecular amplification cycles, 25 - 32 molecular amplification cycles, 25 - 31 molecular amplification cycles, 25 - 30 molecular amplification cycles, 25 - 29 molecular amplification cycles, or 25 - 28 molecular amplification cycles.

[0166] In some embodiments, the molecular amplification product (e.g., a PCR product) is detectable after 25 or more molecular amplification cycles. In some embodiments, the nucleic acid is detectable, for example, after 25 or more molecular amplification cycles, 26 or more molecular amplification cycles, 27 or more molecular amplification cycles, 28 or more molecular amplification cycles, 29 or more molecular amplification cycles, 30 or more molecular amplification cycles, 31 or more molecular amplification cycles, 32 or more molecular amplification cycles, 33 or more molecular amplification cycles, 34 or more molecular amplification cycles, or 35 or more molecular amplification cycles.

[0167] In some embodiments, the molecular amplification product (e.g., a PCR product) is detected after the nucleic acid has been amplified using 10 - 40 molecular amplification cycles, 10 - 35 molecular amplification cycles, 20 - 35 molecular amplification cycles, 25 - 35 molecular amplification cycles, 26 - 35 molecular amplification cycles, 27 - 35 molecular amplification cycles, 28 - 35 molecular amplification cycles, 29 - 35 molecular amplification cycles, or 30 - 35 molecular amplification cycles.

[0168] In some embodiments, the detection of nucleic acid is performed simultaneously with the molecular amplification of nucleic acid. In some embodiments, the detection of nucleic acid and the molecular amplification of nucleic acid are performed sequentially. In some embodiments, the detection of nucleic acid is performed after each cycle of molecular amplification.

[0169] In some embodiments, the method of analyzing nucleic acid further comprises at least one of the following steps: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method of analyzing nucleic acid further comprises at least two of the following steps: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method of analyzing nucleic acid further comprises the steps of detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method of analyzing nucleic acid comprises the step of detecting the nucleic acid by fluorescence detection.

[0170] As described herein, the processed sample to be heated contains at least nucleic acid and, optionally, reagents such as enzymes or chelating agents. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is derived from eukaryotic cells or prokaryotic cells. In some embodiments, the nucleic acid is derived from a virus. In some embodiments, the nucleic acid is selected from viral nucleic acid, bacterial nucleic acid, protozoal nucleic acid, eukaryotic nucleic acid, and fungal nucleic acid. In some embodiments, the nucleic acid is viral nucleic acid.

[0171] In some embodiments, the nucleic acid is extracted from an organism selected from prokaryotes. In some embodiments, the nucleic acid is extracted from an organism selected from eukaryotes. In some embodiments, the nucleic acid is extracted from a parasite. In some embodiments, the nucleic acid is extracted from a virus, bacterium, fungus, animal, or plant.

[0172] In some embodiments, the nucleic acid is extracted from a virus. In some embodiments, the nucleic acid is extracted from a respiratory virus. In some embodiments, the virus is selected from influenza virus, rhinovirus, coronavirus, metapneumovirus, adenovirus, syncytial virus, bocavirus, and parainfluenza virus.

[0173] In some embodiments, the nucleic acid is extracted from bacteria. In some embodiments, the nucleic acid is extracted from gram-negative bacteria. In some embodiments, the nucleic acid is extracted from gram-positive bacteria. In some embodiments, the nucleic acid is extracted from fungi. In some embodiments, the nucleic acid is extracted from yeast. In some embodiments, the nucleic acid is extracted from animals. In some embodiments, the nucleic acid is extracted from plants.

[0174] In some cases, the nucleic acid is in a body sample. In some embodiments, the nucleic acid has not been extracted, isolated, or otherwise purified from the body sample. The body sample may be selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In some embodiments, the body sample comprises a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymph fluid, bile, synovial fluid, cyst fluid, ascites, pleural effusion, ocular fluid, interstitial fluid, endocervical mucus, and urine. In some embodiments, the body sample comprises mucus. In some embodiments, the body sample comprises a body fluid sample, tissue, or cells of a subject.

[0175] In some aspects, the body sample may be collected from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a non-human primate (such as marmoset, macaque, chimpanzee, etc.), a rodent (such as mouse, rat, gerbil, globefish mouse, hamster, cotton mouse, naked mole rat, etc.), a rabbit, a domesticated mammal (such as goat, sheep, pig, dairy cow, bull, horse, camel, etc.), a pet animal (such as dog, cat, etc.), or a zoo mammal. In some embodiments, the subject is a human.

[0176] The bodily sample may be collected from the subject by a nasopharyngeal swab, a cervical swab, or a nasal swab. In some cases, the bodily sample may contain a pathogen or a part thereof. In some embodiments, the pathogen or a part thereof is selected from a virus or a part thereof, a bacterium or a part thereof, a protozoan or a part thereof, a yeast or a part thereof, and a fungus or a part thereof. In some embodiments, the pathogen is a blood-bone pathogen or a part thereof. In some embodiments, the pathogen is a respiratory pathogen or a part thereof. In some embodiments, the respiratory pathogen includes a bacterium or a fungal pathogen. In some embodiments, the respiratory pathogen is Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, Klebsiella pneumoniae, Staphylococcus aureus, or Aspergillus sp. In some embodiments, the pathogen is a virus or a part thereof. In some embodiments, the virus is a respiratory virus. In some embodiments, the virus is SARS-CoV2, influenza A virus (-H1N1 and other subtypes), influenza B virus, human respiratory syncytial virus (HRSV), type I (HPIV-1), type II (HPIV-2), type III (HPIV-3), type IV (HPIV-4) human parainfluenza virus, rhinovirus / enterovirus (RV / EV), adenovirus (ADV), human metapneumovirus (hMPV), human coronavirus (HCoV)-229E, HCoV-HKU1, HCoV-NL63, or HCoV-OC43. In some embodiments, the virus is SARS-CoV2.

[0177] In some embodiments, the virus is selected from influenza virus, rhinovirus, coronavirus, metapneumovirus, adenovirus, syncytial virus, bocavirus, and parainfluenza virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is rhinovirus. In some embodiments, the virus is coronavirus. In some embodiments, the virus is metapneumovirus. In some embodiments, the virus is adenovirus. In some embodiments, the virus is syncytial virus. In some embodiments, the virus is bocavirus. In some embodiments, the virus is parainfluenza virus.

[0178] In some aspects, the processed sample includes a bodily sample and is heated in a closed heating chamber under high pressure conditions to a temperature above 100 degrees Celsius, thereby generating a heat-treated sample.

[0179] In some aspects, the processed sample includes cells containing nucleic acids. The cells may be non-lysed cells. The cells may be eukaryotic or prokaryotic cells. In some embodiments, the cells are bacterial cells, such as B. subtilis, E. Coli, or S. Pyogenes. In some embodiments, the cells are fungal cells, such as C. albicans. In some embodiments, the cells are yeast cells, such as S. cerevisiae. In some embodiments, the cells are mammalian cells, such as Chinese hamster ovary cells, BHK cells, or mouse C127 cells. In some embodiments, the cells are human cells, such as HeLa cells. In some embodiments, the cells are bacterial spores, such as B. cereus bacterial spores.

[0180] In some embodiments, the cells may be in a bodily sample. In some embodiments, the cells are embedded in a biological matrix, such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine, or saliva. In some embodiments, the cells are embedded in nasal mucus.

[0181] The processed sample may be an aqueous solution or suspension. In some embodiments, the processed sample comprises isolated nucleic acids diluted in a collection buffer. In other embodiments, the processed sample comprises a bodily sample (e.g., saliva or mucus) containing nucleic acids diluted in a collection buffer. In further embodiments, the processed sample comprises a bodily sample containing nucleic acids that is not diluted. In some embodiments, the processed sample comprises cells suspended in a collection buffer. The collection solution or buffer in the present disclosure can be made of pure water or can be a mix of low buffering capacity buffers. Examples of such buffers can be based on Tris HCl buffer in the range of 1 mM to 50 mM with or without EDTA at a concentration in the range of 0.5 mM to 1 mM.

[0182] In some embodiments, the processed sample has a pH of from about 8.0 to about 12.0. In some embodiments, the processed sample has a pH of from about 9.0 to about 12.0. In some embodiments, the processed sample has a pH of from about 10.0 to about 12.0. In some embodiments, the processed sample has a pH of from about 11.0 to about 12.0. In some embodiments, the processed sample has a pH of from about 8.0 to about 11.0. In some embodiments, the processed sample has a pH of from about 9.0 to about 11.0. In some embodiments, the processed sample has a pH of from about 10.0 to about 11.0. In some embodiments, the processed sample has a pH of from about 8.0 to about 10.0. In some embodiments, the processed sample has a pH of from about 9.0 to about 10.0.

[0183] In some embodiments, the processed sample has a pH greater than 7.0. In some embodiments, the processed sample has a pH greater than 8.0. In some embodiments, the processed sample has a pH greater than 8.5. In some embodiments, the processed sample has a pH greater than 9.0. In some embodiments, the processed sample has a pH greater than 9.5. In some embodiments, the processed sample has a pH greater than 10.0. In some embodiments, the processed sample has a pH greater than 10.5. In some embodiments, the processed sample has a pH greater than 11.0.

[0184] In some embodiments, the processed sample has a pH of about 7.0. In some embodiments, the processed sample has a pH of about 8.0. In some embodiments, the processed sample has a pH of about 9.0. In some embodiments, the processed sample has a pH of about 10.0. In some embodiments, the processed sample has a pH of about 11.0. In some embodiments, the processed sample has a pH of about 12.0.

[0185] In some embodiments, the processed sample has a pH of about 8.0 to 11.0. In some embodiments, the processed sample has a pH of about 9.0 to 11.0. In some embodiments, the processed sample has a pH of about 10.0 to 11.0.

[0186] In some embodiments, the processed sample has a pH of about 8.0 to 12.0. In some embodiments, the processed sample has a pH of about 9.0 to 12.0. In some embodiments, the processed sample has a pH of about 10.0 to 12.0. In some embodiments, the processed sample has a pH of about 11.0 to 12.0.

[0187] In some embodiments, the processed sample has a pH of about 4.0 to about 7.0. In some embodiments, the processed sample has a pH of about 5.0 to about 7.0. In some embodiments, the processed sample has a pH of about 6.0 to about 7.0. In some embodiments, the processed sample has a pH of about 4.0 to about 6.0. In some embodiments, the processed sample has a pH of about 5.0 to about 6.0. In some embodiments, the processed sample has a pH of about 4.0 to about 5.0.

[0188] In some embodiments, the processed sample has a pH of about 3.0. In some embodiments, the processed sample has a pH of about 4.0. In some embodiments, the processed sample has a pH of about 5.0. In some embodiments, the processed sample has a pH of about 6.0. In some embodiments, the processed sample has a pH of about 7.0.

[0189] In some embodiments, the processed sample has a pH below about 3.0. In some embodiments, the processed sample has a pH below about 4.0. In some embodiments, the processed sample has a pH below about 5.0. In some embodiments, the processed sample has a pH below about 6.0. In some embodiments, the processed sample has a pH below about 7.0.

[0190] In some embodiments, the volume of the processed sample is the total volume of the mixture of the biological sample and collection buffer with respect to one or more additives before high-temperature heating. In some embodiments, the volume of the processed sample is from 100 μL to 5 mL. In some embodiments, the volume of the processed sample is from 200 μL to 5 mL. In some embodiments, the volume of the processed sample is from 300 μL to 5 mL. In some embodiments, the volume of the processed sample is from 400 μL to 5 mL. In some embodiments, the volume of the processed sample is from 500 μL to 5 mL. In some embodiments, the volume of the processed sample is from 1 mL to 5 mL. In some embodiments, the volume of the processed sample is from 1.5 mL to 5 mL. In some embodiments, the volume of the processed sample is from 2 mL to 5 mL. In some embodiments, the volume of the processed sample is from 2.5 mL to 5 mL. In some embodiments, the volume of the processed sample is from 3 mL to 5 mL. In some embodiments, the volume of the processed sample is from 3.5 mL to 5 mL. In some embodiments, the volume of the processed sample is from 4 mL to 5 mL. In some embodiments, the volume of the processed sample is from 4.5 mL to 5 mL.

[0191] In some embodiments, the processed sample contains nucleic acids at 10 copies / mL to 10 9 copies / mL. In some embodiments, the processed sample contains nucleic acids at 50 copies / mL to 10 9 copies / mL. In some embodiments, the processed sample contains nucleic acids at 100 copies / mL to 10 9 copies / mL. In some embodiments, the processed sample contains nucleic acids at 10 3 copies / mL to 10 9 copies / mL. In some embodiments, the processed sample contains nucleic acids at 10 4 copies / mL to 10 9contains nucleic acid at 5 copies / mL. In some embodiments, the processed sample is 10 9 copies / mL to 10 6 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 9 copies / mL to 10

[0192] In some embodiments, the processed sample is 10 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 50 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 100 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 3 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 4 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 5 copies / mL to 10 8 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 6 copies / mL to 10 8 copies / mL of nucleic acid.

[0193] In some embodiments, the processed sample is 10 copies / mL to 10 7 copies / mL of nucleic acid. In some embodiments, the processed sample is 50 copies / mL to 10 7 copies / mL of nucleic acid. In some embodiments, the processed sample is 100 copies / mL to 10 7 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 3 copies / mL to 10 7 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 4 copies / mL to 10 7 copies / mL of nucleic acid. In some embodiments, the processed sample is 10 5 copies / mL to 10 7It contains nucleic acid at 6 copies / mL to 7 copies / mL of nucleic acid.

[0194] In some embodiments, the processed sample contains one or more additives. The one or more additives may include a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, a protease, a nuclease inhibitor, or a combination thereof.

[0195] In some embodiments, the processed sample contains a chelating agent. In some embodiments, the chelating agent is an insoluble chelating agent. In some embodiments, the insoluble chelating agent includes a chelating resin. The chelating resin may be a polymer or a copolymer. The chelating resin may be a cation binder or a metal ion binder. The chelating resin may be in the form of microbeads. In some embodiments, the chelating agent includes cross-linked polystyrene. The chelating agent may include one or more functional groups. The one or more functional groups may include a sulfonic acid or sulfonate group; a quaternary amino group (e.g., trimethylammonium); a primary, secondary, and / or tertiary amino group (e.g., polyethyleneamine); or a carboxylic acid or carboxylate group. In some embodiments, the insoluble chelating agent includes a styrene divinylbenzene copolymer. In some embodiments, the insoluble chelating agent includes Chelex resin or Chelex. In some embodiments, Chelex is stored in the collection buffer.

[0196] In some embodiments, the chelating agent is a soluble chelating agent. In some embodiments, the soluble chelating agent includes ethylenediaminetetraacetic acid (EDTA). In some embodiments, the chelating agent includes EDTA, nitrilotriacetic acid, n-hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediamine, dimercaprol, porphyrin, heme, hemoglobin, or chlorophyll. In some embodiments, the chelating agent includes simple organic acids such as oxalic acid, malic acid, rubic acid, or citric acid.

[0197] In some embodiments, the chelating agent is added at a final concentration defined as the weight / volume percentage of the weight of the chelating agent to the volume of the processed sample (e.g., a mixture containing a biological sample, a collection buffer, a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent) before the heating step. In some embodiments, the final concentration of the chelating agent in the processed sample before the heating step is 1 weight / volume% to 40 weight / volume%, 2.5 weight / volume% to 35 weight / volume%, 5 weight / volume% to 25 weight / volume%, 7.5 weight / volume% to 20 weight / volume%, or 10 weight / volume% to 15 weight / volume% of the weight of the chelating agent to the volume of the processed sample. In some embodiments, the final concentration of the chelating agent in the processed sample before the heating step is about 2 weight / volume%, about 4 weight / volume%, about 6 weight / volume%, about 8 weight / volume%, about 10 weight / volume%, about 12 weight / volume%, about 14 weight / volume%, about 16 weight / volume%, about 18 weight / volume%, or about 20 weight / volume% of the weight of the chelating agent to the volume of the processed sample.

[0198] In some embodiments, the processing sample further comprises a single-stranded nucleic acid binding (SSB) protein. In some embodiments, the single-stranded nucleic acid binding protein is thermostable. In some embodiments, the SSB protein is thermostable at a temperature of 4°C to 170°C, 4°C to 160°C, 4°C to 150°C, 4°C to 140°C, 4°C to 130°C, or 4°C to 120°C. In some embodiments, the SSB protein is thermostable at a temperature of 90°C to 170°C, 90°C to 160°C, 90°C to 150°C, 90°C to 140°C, 90°C to 130°C, 90°C to 120°C, or 90°C to 110°C.

[0199] In some embodiments, the final molar concentration of the SSB protein in the processing sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, an SSB protein, and a reducing agent) before the heating step is a concentration of 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.2 μM to 0.9 μM, 0.3 μM to 0.7 μM, or 0.4 μM to 0.6 μM.

[0200] In some embodiments, the final molar concentration of the SSB protein in the processing sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, an SSB protein, and a reducing agent) before the heating step is about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.2 μM, about 2.4 μM, 2.6 μM, about 2.8 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM.

[0201] In some embodiments, the single-stranded nucleic acid binding protein is derived from a thermophilic organism. In some embodiments, the thermophilic organism is a thermophilic microorganism or a thermophilic bacterium. In some embodiments, the single-stranded nucleic acid binding protein is derived from an organism selected from Thermotoga maritima (TmaSSB), Thermotoga neapolitana (TneSSB), Thermococcus kodakarensis (KOD), and Thermus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid binding protein is derived from, for example, Thermus aquaticus (TaqSSB) or Thermococcus kodakarensis (KOD). In some embodiments, the single-stranded nucleic acid binding protein is derived from an organism selected from Thermotoga maritima (TmaSSB), Thermotoga neapolitana (TneSSB), and Thermus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid binding protein is derived from Thermotoga maritima (TmaSSB). In some embodiments, the single-stranded nucleic acid binding protein is derived from Thermotoga neapolitana (TneSSB). In some embodiments, the single-stranded nucleic acid binding protein is derived from Thermococcus kodakarensis (KOD). In some embodiments, the single-stranded nucleic acid binding protein is derived from Thermus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid binding protein is selected from ET SSB, E.Coli SSB, KOD SSB, TthSSB, TneSSB, TmaSSB, and TaqSSB.

[0202] In some embodiments, the processed sample contains a reducing agent. In some embodiments, the reducing agent is added to the processed sample before the heating step. The reducing agent may be 2-mercaptoethanol, 2-mercaptoethylamine-HCl, TCEP, cysteine-HCl, dithiothreitol (DTT), TCEP-HCl, a thiol-based reducing agent, guanidine-HCl, or urea.

[0203] In some embodiments, the final molar concentration of the reducing agent in the processed sample (e.g., a mixture containing a biological sample, a collection buffer, a chelating agent, an SSB protein, and a reducing agent) before the heating step is from 0.1 mM to 10 mM, from 0.1 mM to 9 mM, from 0.1 mM to 8 mM, from 0.1 mM to 7 mM, from 0.1 mM to 6 mM, from 0.1 mM to 5 mM, from 0.1 mM to 4 mM, from 0.1 mM to 3 mM, from 0.1 mM to 2 mM, from 0.2 mM to 1.8 mM, from 0.4 mM to 1.6 mM, from 0.6 mM to 1.4 mM, or from 0.8 mM to 1.2 mM. In some embodiments, the final molar concentration of the reducing agent in the processed sample (e.g., a mixture containing a biological sample, a collection buffer, a chelating agent, an SSB protein, and a reducing agent) before the heating step is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 1.2 mM, about 1.4 mM, about 1.6 mM, about 1.8 mM, about 2 mM, about 2.5 mM, about 5 mM, about 7.5 mM, or about 10 mM.

[0204] In some embodiments, the processed sample comprises one or more reagents selected from a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the processed sample comprises at least two reagents selected from a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the processed sample comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the concentration of the chelating agent is 2.5 wt% to 35 wt% of the processed sample, the concentration of the single-stranded nucleic acid-binding protein in the processed sample is 0.1 μM to 5 μM, and the concentration of the reducing agent in the processed sample is 0.1 mM to 5 mM. In some embodiments, the concentration of the chelating agent is 5 wt% to 25 wt% of the processed sample, the concentration of the single-stranded nucleic acid-binding protein in the processed sample is 0.1 μM to 2 μM, and the concentration of the reducing agent in the processed sample is 0.1 mM to 2 mM. In some embodiments, the concentration of the chelating agent is 10 wt% to 15 wt% of the processed sample, the concentration of the single-stranded nucleic acid-binding protein in the processed sample is 0.3 μM to 0.7 μM, and the concentration of the reducing agent in the processed sample is 0.6 mM to 1.4 mM.

[0205] In some embodiments, the processed sample contains a stabilizer. In some embodiments, the processed sample is mixed with a stabilizer prior to the heating step. In some cases, the stabilizer may help prevent the degradation of nucleic acids. In some cases, the stabilizer may help inactivate molecular amplification inhibitors during high-pressure heating. In some embodiments, the stabilizer may stabilize proteins. In some embodiments, the stabilizer may stabilize nucleic acids. In some embodiments, the stabilizer may stabilize both nucleic acids and proteins. The stabilizer may improve the viscosity of the processed sample. The stabilizer may help prevent or reduce aggregation between one or more proteins. The stabilizer may improve the solubility of proteins. The stabilizer may reduce non-specific binding between one or more components (e.g., proteins). The stabilizer may be a blocker. The stabilizer may be a component that stabilizes one or more components in the processed sample. In some embodiments, the stabilizer may stabilize nucleic acids. In some embodiments, the stabilizer may stabilize chelating agents. In some embodiments, the stabilizer may stabilize single-stranded nucleic acid-binding proteins. For example, the stabilizer may be bovine serum albumin (BSA). The stabilizer may be gelatin. In some embodiments, the stabilizer has a concentration in the processed sample of 100 ng / mL to 15 mg / mL, 200 ng / mL to 15 mg / mL, 300 ng / mL to 15 mg / mL, 400 ng / mL to 15 mg / mL, 500 ng / mL to 15 mg / mL, or 1 mg / mL to 15 mg / mL. In some embodiments, the stabilizer has a concentration in the processed sample of 100 ng / mL to 10 mg / mL, 200 ng / mL to 10 mg / mL, 300 ng / mL to 10 mg / mL, 400 ng / mL to 10 mg / mL, 500 ng / mL to 10 mg / mL, or 1 mg / mL to 10 mg / mL. In some embodiments, the stabilizer has a concentration in the processed sample of 500 ng / mL to 2 mg / mL, 500 ng / mL to 3 mg / mL, 500 ng / mL to 4 mg / mL, 500 ng / mL to 5 mg / mL, 500 ng / mL to 6 mg / mL, 500 ng / mL to 7 mg / mL, 500 ng / mL to 8 mg / mL, 500 ng / mL to 9 mg / mL, or 500 ng / mL to 10 mg / mL.

[0206] In some embodiments, the processed sample comprises one or more reagents selected from chelating agents, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the processed sample comprises at least two reagents selected from chelating agents, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the processed sample comprises at least three reagents selected from chelating agents, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the processed sample comprises a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent. In some embodiments, the concentration of the chelating agent is 2.5% to 35% by weight / volume of the processed sample, the concentration of the single-stranded nucleic acid binding protein in the processed sample is 0.1 μM to 5 μM, the concentration of the reducing agent in the processed sample is 0.1 mM to 5 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL. In some embodiments, the concentration of the chelating agent is 5% to 25% by weight / volume of the processed sample, the concentration of the single-stranded nucleic acid binding protein in the processed sample is 0.1 μM to 2 μM, the concentration of the reducing agent in the processed sample is 0.1 mM to 2 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL. In some embodiments, the concentration of the chelating agent is 10% to 15% by weight / volume of the processed sample, the concentration of the single-stranded nucleic acid binding protein in the processed sample is 0.3 μM to 0.7 μM, the concentration of the reducing agent in the processed sample is 0.6 mM to 1.4 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL.

[0207] In some embodiments, the processed sample contains protease. In some embodiments, the processed sample is mixed with protease before the heating step. In some embodiments, the protease is, for example, proteinase K. In some embodiments, the biological sample is mixed with a reducing agent and protease before the heating step. In some embodiments, the final concentration of protease in the processed sample (e.g., a mixture containing a biological sample, collection buffer, chelating agent, SSB protein, reducing agent, and protease) before the heating step is a concentration of 0.01 mg / mL to 5 mg / mL, 0.02 mg / mL to 4 mg / mL, 0.03 mg / mL to 3 mg / mL, 0.04 mg / mL to 2 mg / mL, 0.05 mg / mL to 1 mg / mL, 0.075 mg / mL to 0.75 mg / mL, or 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the final concentration of protease in the processed sample (e.g., a mixture containing a biological sample, collection buffer, chelating agent, SSB protein, reducing agent, and protease) before the heating step is about 0.05 mg / mL, about 0.075 mg / mL, about 0.1 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, or about 1 mg / mL.

[0208] In some embodiments, the processed sample contains a nuclease inhibitor. In some embodiments, the processed sample contains an RNAse inhibitor. In some embodiments, an amount of RNAse inhibitor, for example, about 1 U, about 10 U, about 50 U, about 100 U, about 150 U, about 200 U, about 250 U, about 300 U, about 350 U, about 400 U, about 450 U, or about 500 U, is added before the heating step.

[0209] In some embodiments, the amount of RNase inhibitor in the processed sample is from about 1 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 10 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 50 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 100 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 150 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 200 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 250 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 300 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 350 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 400 U to about 500 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 450 U to about 500 U.

[0210] In some embodiments, the amount of RNase inhibitor in the processed sample is from about 1 U to about 200 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 10 U to about 200 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 50 U to about 200 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 100 U to about 200 U. In some embodiments, the amount of RNase inhibitor in the processed sample is from about 150 U to about 200 U.

[0211] In some embodiments, the processed sample further comprises a molecular amplification inhibitor. In some cases, the molecular amplification inhibitor may be an agent that binds to nucleic acids. The molecular amplification inhibitor may be an agent that degrades nucleic acids. In some cases, the molecular amplification inhibitor may be a nuclease. In some cases, the molecular amplification inhibitor is a DNase. In other cases, the molecular amplification inhibitor is an RNase. In some embodiments, the processed sample comprises one or more molecular amplification inhibitors that can be inactivated after the heating step.

[0212] As described herein, the processing sample may be heated in a closed heating chamber. The closed heating chamber may substantially prevent air and vapor from entering or exiting the chamber. In some cases, there may be a negligible air flow entering and leaving the closed heating chamber. The closed heating chamber may remain closed during high-pressure heating. In some embodiments, the closed heating chamber is a chamber inside the heating vessel. The heating vessel and / or the closed heating chamber may be composed of different materials. In some embodiments, the heating vessel and / or the closed heating chamber include glass. In some embodiments, the heating vessel and / or the closed heating chamber include high-temperature polycarbonate. In some embodiments, the heating vessel and / or the closed heating chamber include a thermally conductive material. In some embodiments, the heating vessel and / or the closed heating chamber include metal. In some embodiments, the heating vessel and / or the closed heating chamber may include zinc, stainless steel, copper, copper alloy, gold, silver, aluminum, aluminum nitride, iron, nickel, nickel alloy, cobalt, carbon fiber, platinum, brass, tungsten, silicon, silicon carbide, diamond, or graphite. In some embodiments, the heating vessel and / or the closed heating chamber include a conductive material. In some embodiments, the heating vessel and / or the closed heating chamber include ferromagnetic materials such as iron, nickel, and cobalt. For example, the heating vessel and / or the closed heating chamber may be or include a glass ampoule, a plastic container, or a metal container. In some embodiments, the heating vessel and / or the closed heating chamber include an induction susceptor 103 (e.g., a metal cup) (Figs. 6, 7, 9) that can be used to heat the processing sample by induction heating due to contact with the induction platform 801 (Fig. 8).

[0213] In some embodiments, the heating vessel includes a heating chamber 702 that contains the processing sample during high-pressure heating (Figure 7). The material of the vessel and / or the heating chamber may be selected such that the heating chamber can have a temperature gradient rate of 2 degrees Celsius per second to 50 degrees Celsius per second, 2 degrees Celsius per second to 40 degrees Celsius per second, 2 degrees Celsius per second to 35 degrees Celsius per second, 2 degrees Celsius per second to 30 degrees Celsius per second, 2 degrees Celsius per second to 25 degrees Celsius per second, 2 degrees Celsius per second to 20 degrees Celsius per second, or 2 degrees Celsius per second to 15 degrees Celsius per second. The material of the vessel and / or the heating chamber may be selected such that the heating chamber can have a temperature gradient rate of 5 degrees Celsius per second to 60 degrees Celsius per second, 5 degrees Celsius per second to 50 degrees Celsius per second, 5 degrees Celsius per second to 40 degrees Celsius per second, 5 degrees Celsius per second to 35 degrees Celsius per second, 5 degrees Celsius per second to 30 degrees Celsius per second, 5 degrees Celsius per second to 25 degrees Celsius per second, 5 degrees Celsius per second to 20 degrees Celsius per second, or 5 degrees Celsius per second to 15 degrees Celsius per second.

[0214] In some embodiments, the heating chamber is closed during high-pressure heating to prevent air and vapor from entering or exiting the chamber. In some embodiments, the closed heating chamber remains closed under high-pressure conditions, and the pressure inside the chamber is higher than the pressure outside the chamber. The material of the container or the heating chamber may be selected such that the closed heating chamber can remain closed, for example, at room temperature to 160 degrees Celsius, or during high-pressure heating when the pressure inside the heating chamber is 1 to 200 PSI relative to 1 atm.

[0215] In some embodiments, the closed heating chamber may be or include a glass ampoule. The glass ampoule may include a sample inlet that is fused. In other embodiments, the closed heating chamber may be or include a plastic container. The plastic container may include a heat-resistant plastic, such as a plastic resin, or polycarbonate, high-density polypropylene, PEEK, or PEI. For example, the plastic container may be an ultra-low temperature tube or a part of a microfluidic cartridge. In further embodiments, the closed heating chamber may be or include a metal container. The metal container may include an induction susceptor (e.g., a metal cup).

[0216] In some embodiments, the closed heating chamber is produced by sealing an open heating chamber. The sealing operation may include fusing an open sample inlet. The sealing operation may include using a cap, lid, plug, or valve. The manner of sealing the heating chamber may be selected such that the closed heating chamber can remain closed during high-pressure heating.

[0217] In some embodiments, the heating vessel includes a sample inlet 101 through which a sample can enter the heating chamber 102, as shown in FIGS. 6, 7, 9, and 11. The sample inlet can be sealed to create a closed heating chamber within the heating vessel. In some embodiments, the sample inlet is sealed using a cap, lid, plug, or valve. In some embodiments, the sample inlet may include a valve. The valve may be a one-way valve 201 (e.g., a duckbill valve) that allows the sample to enter the heating chamber and prevents air and / or the sample from exiting the heating chamber (FIGS. 7A, 7B). In some cases, the sample is introduced through the one-way valve 201 (FIGS. 7A, 7B). The sample may be manually introduced using a pipette or syringe needle. In other cases, the sample may be introduced by an automated system. The heating vessel may be part of a fluid system, and the one-way valve may be in contact with components upstream of the fluid system. In some embodiments, the one-way valve can further prevent air and vapor from exiting the heating chamber. The one-way valve provides a seal 202 for the closed chamber during high-pressure heating (FIG. 7).

[0218] In some embodiments, the closed chamber is opened at some point to release the sample from the heating vessel. The closed chamber may be opened after high-pressure heating. In some embodiments, the heating chamber is opened using mechanical force. In some embodiments, the heating chamber is opened by removing a cap, lid, plug, or valve from the outlet. In some embodiments, the heating chamber is opened by opening the discharge valve 302 at the outlet. For example, the discharge valve 302 may be a laser valve that is initially closed and opened by shining a laser beam on the valve (FIGS. 10, 11). In some cases, the outlet may be the same as or in the same location as the sample inlet. In other cases, the outlet is in a different location from the sample inlet.

[0219] In some embodiments, the sample can be released from the outlet. The outlet can be a portion of the outlet channel 301 within the heating vessel (Figs. 9, 10). The outlet channel 301 can include or be in contact with the discharge valve 302 (Figs. 9, 10). The outlet channel 301 can initially be sealed by the discharge valve 302, for example, during sample injection in operation 1101 of Fig. 11A and during high-pressure heating. The outlet channel 301 can then be unsealed by opening the discharge valve 302, for example, for sample release in operation 1102 of Fig. 11B. When the discharge valve 302 is opened, the sample can be discharged from the heating chamber through the outlet channel 301. The sample can be pushed out of the heating chamber through the outlet channel 301 using a force, for example, centrifugal force. In some embodiments, the sample is discharged into a collection chamber 303 (Fig. 9). In some embodiments, the collection chamber is part of the heating vessel. In some embodiments, the collection chamber and the heating vessel are part of a fluid network.

[0220] After release from the high-pressure heating vessel, the processed sample after high-pressure heating can undergo further sample processing or analysis. The processed sample after high-pressure heating can be used for nucleic acid detection and / or analysis using any of PCR analysis or biological (e.g., diagnostic) assays, or nucleic acid analysis methods disclosed elsewhere in this specification.

[0221] Certain embodiments of the present disclosure

[0222] Embodiment 1. A method for analyzing nucleic acids in a biological sample, comprising heating the biological sample to a temperature above the boiling point of the biological sample in a closed container, and analyzing the nucleic acids.

[0223] Embodiment 2. A method for analyzing nucleic acids in a biological sample, comprising heating the biological sample to a temperature of at least 101 degrees Celsius in a closed container, and analyzing the nucleic acids.

[0224] Embodiment 3. A method for analyzing nucleic acids in a biological sample, comprising the steps of heating a biological sample containing nucleotides in a closed container, thereby generating an internal pressure of the container between 1 and 200 PSI with respect to the atmosphere, and analyzing the nucleic acids.

[0225] Embodiment 4. A method for analyzing nucleic acids in a biological sample, comprising the steps of heating the biological sample under high pressure conditions and analyzing the nucleic acids.

[0226] Embodiment 5. A method for improving the efficiency of molecular amplification, comprising the step of heating a biological sample in a closed container, thereby generating an internal pressure of the container between 10 and 100 PSI with respect to the atmosphere, wherein the biological sample contains nucleic acids.

[0227] Embodiment 6. A method for inactivating a molecular amplification inhibitor in a biological sample containing nucleic acids, comprising the step of heating the biological sample to a temperature above the boiling point of the biological sample, wherein the nucleic acids are not substantially degraded.

[0228] Embodiment 7. The method according to Embodiment 5 or Embodiment 6, further comprising the step of analyzing the nucleic acids.

[0229] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the step of analyzing the nucleic acids comprises one or more of the following: detecting the nucleic acids; sequencing the nucleic acids; and genotyping the nucleic acids.

[0230] Embodiment 9. The method according to any one of Embodiments 1 to 8, further comprising the step of detecting the nucleic acids by fluorescence detection.

[0231] Embodiment 10. The method according to any one of Embodiments 1 to 9, further comprising the step of amplifying the nucleic acids using molecular amplification.

[0232] Embodiment 11. The method according to Embodiment 10, further comprising the step of amplifying the nucleic acids using molecular amplification before the step of analyzing the nucleic acids.

[0233] Embodiment 12. The method according to Embodiment 11, further comprising a step of amplifying nucleic acid using molecular amplification after the step of heating the biological sample.

[0234] Embodiment 13. A method for analyzing nucleic acid in a biological sample, comprising: a step of heating the biological sample in a closed container; a step of generating a pressure exceeding 1 atm in the closed container; a step of amplifying nucleic acid using molecular amplification; and a step of analyzing the nucleic acid.

[0235] Embodiment 14. A method for analyzing nucleic acid in a biological sample, comprising: a step of heating the biological sample to a temperature exceeding the boiling point of the biological sample under high-pressure conditions; a step of amplifying nucleic acid using molecular amplification; and a step of analyzing the nucleic acid.

[0236] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the heating of the biological sample occurs during a first period.

[0237] Embodiment 16. The method according to Embodiment 15, wherein the first period is between 30 seconds and 3 minutes.

[0238] Embodiment 17. The method according to any one of Embodiments 10 to 16, wherein the molecular amplification includes polymerase chain reaction ("PCR").

[0239] Embodiment 18. The method according to Embodiment 17, wherein the analysis of the nucleic acid includes detection of a PCR product formed during the molecular amplification.

[0240] Embodiment 19. The method according to any one of Embodiments 1 to 18, wherein the biological sample further contains an additive.

[0241] Embodiment 20. The method according to Embodiment 19, wherein the additive is selected from a chelating agent, a reducing agent, or a nuclease inhibitor.

[0242] Embodiment 21. The method according to embodiment 20, wherein the additive comprises both a chelating agent and a reducing agent.

[0243] Embodiment 22. The method according to embodiment 19 or embodiment 20, wherein the additive is a chelating agent.

[0244] Embodiment 23. The method according to any one of embodiments 20 to 22, wherein the chelating agent is an insoluble chelating agent.

[0245] Embodiment 24. The method according to embodiment 23, wherein the insoluble chelating agent comprises Chelex.

[0246] Embodiment 25. The method according to any one of embodiments 20 to 22, wherein the chelating agent is a soluble chelating agent.

[0247] Embodiment 26. The method according to embodiment 25, wherein the soluble chelating agent comprises EDTA.

[0248] Embodiment 27. The method according to embodiment 19 or embodiment 20, wherein the additive is a reducing agent.

[0249] Embodiment 28. The method according to any one of embodiments 19 to 27, wherein the additive is added to the biological sample before the heating step.

[0250] Embodiment 29. The method according to any one of embodiments 1 to 28, wherein the biological sample further comprises a single-stranded binding protein.

[0251] Embodiment 30. The method according to any one of embodiments 1 to 29, wherein the nucleic acid is analyzed after being amplified using a molecular amplification cycle between 10 and 45.

[0252] Embodiment 31. The method according to any one of embodiments 1 to 30, wherein the nucleic acid is detectable after a smaller number of molecular amplification cycles than required in the absence of heating.

[0253] Embodiment 32. The method according to any one of Embodiments 1 to 31, wherein the nucleic acid is detectable after a smaller number of molecular amplification cycles than required as compared to heating the sample at a temperature below the boiling point of the sample for the same time.

[0254] Embodiment 33. The method according to any one of Embodiments 1 to 32, wherein the nucleic acid is detectable after 20 to 45 molecular amplification cycles.

[0255] Embodiment 34. The method according to Embodiment 33, wherein the nucleic acid is detectable after 28 to 35 molecular amplification cycles.

[0256] Embodiment 35. The method according to any one of Embodiments 1 to 34, wherein the analysis of the nucleic acid is performed simultaneously with the molecular amplification of the nucleic acid.

[0257] Embodiment 36. The method according to any one of Embodiments 1 to 34, wherein the analysis of the nucleic acid and the molecular amplification of the nucleic acid are performed sequentially.

[0258] Embodiment 37. The method according to any one of Embodiments 1 to 35, wherein the analysis of the nucleic acid is performed after each cycle of molecular amplification.

[0259] Embodiment 38. The method according to any one of Embodiments 1 to 37, wherein the biological sample further comprises a plurality of molecular amplification inhibitors, and at least 70% of the plurality of molecular amplification inhibitors are inactivated after the heating step.

[0260] Embodiment 39. The method according to any one of Embodiments 1 to 37, wherein the biological sample further comprises a plurality of molecular amplification inhibitors, and at least 90% of the plurality of molecular amplification inhibitors are inactivated after the heating step.

[0261] Embodiment 40. The method according to any one of Embodiments 1 to 39, wherein the nucleic acid is not substantially degraded after the heating step.

[0262] Method according to any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is at least 120 degrees Celsius.

[0263] Method according to any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is between 101 degrees Celsius and 160 degrees Celsius.

[0264] Method according to any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is between 120 degrees Celsius and 140 degrees Celsius.

[0265] Method according to any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is about 130 degrees Celsius.

[0266] Method according to any one of Embodiments 1 to 44, wherein the biological sample is heated in an airtight sealed container.

[0267] Method according to Embodiment 45, wherein heating of the biological sample generates a pressure between 10 and 100 PSI inside the airtight sealed container.

[0268] Method according to any one of Embodiments 1 to 46, wherein the biological sample contains a pH of about 8.0 to about 12.0.

[0269] Method according to any one of Embodiments 1 to 47, wherein the nucleic acid is DNA.

[0270] Method according to any one of Embodiments 1 to 47, wherein the nucleic acid is RNA.

[0271] Method according to any one of Embodiments 1 to 49, wherein the nucleic acid is selected from viral nucleic acid, bacterial nucleic acid, protozoal nucleic acid, and fungal nucleic acid.

[0272] Method according to Embodiment 50, wherein the nucleic acid is viral nucleic acid.

[0273] Embodiment 52. The method according to any one of Embodiments 1 to 51, wherein the biological sample contains nucleic acids between 50 copies / mL and 10 9 copies / mL.

[0274] Embodiment 53. The method according to any one of Embodiments 1 to 52, wherein the volume of the biological sample is between 100 μL and 5 mL.

[0275] Embodiment 54. The method according to any one of Embodiments 1 to 53, wherein the biological sample contains a pathogen or a part thereof.

[0276] Embodiment 55. The method according to Embodiment 54, wherein the pathogen or a part thereof is selected from a virus or a part thereof, a bacterium or a part thereof, a protozoan or a part thereof, a yeast or a part thereof, and a fungus or a part thereof.

[0277] Embodiment 56. The method according to Embodiment 55, wherein the pathogen is a virus or a part thereof.

[0278] Embodiment 57. The method according to Embodiment 56, wherein the virus is SARS-CoV2.

[0279] Embodiment 58. The method according to any one of Embodiments 1 to 57, wherein the biological sample contains a substance selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine.

[0280] Embodiment 59. The method according to Embodiment 58, wherein the biological sample contains mucus.

[0281] Embodiment 60. The method according to Embodiment 59, wherein the biological sample is collected from the subject by a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0282] Embodiment 61. The method according to any one of Embodiments 1 to 60, wherein the biological sample is heated by a heat source.

[0283] Embodiment 62. The method according to Embodiment 61, wherein the heat source includes an induction heater, a heating element, or microwaves.

[0284] Embodiment 63. The method according to any one of Embodiments 1 to 2, 6 to 12, or 14 to 62, wherein the biological sample remains at its temperature for between 1 second and 300 seconds.

[0285] Embodiment 64. The method according to any one of Embodiments 1 to 2, 6 to 12, or 14 to 62, wherein the biological sample remains at its temperature for between 30 seconds and 120 seconds.

[0286] Embodiment 65. The method according to any one of Embodiments 1 to 64, wherein the biological sample is collected from a subject.

[0287] Embodiment 66. The method according to Embodiment 65, wherein the subject is a human.

[0288] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

Examples

[0289] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. (Example 1) The high-pressure heating method effectively neutralizes RT-PCR inhibitors

[0290] In this example, samples for RT-PCR were prepared using the methods of the present disclosure and these methods were compared with standard methods. The efficiency of RT-PCR (represented by the average cycle threshold) from two methods of sample preparation was compared.

[0291] Materials Used Ultra-low temperature tube; 6mm disk of ferromagnetic zinc coating made of metal; 1.5ml collection buffer composed of 10mm Tris / EDTA and 1mM NaCl; 10% W / v Chelex 100 resin; CDC SARS-COV2 RT-PCR primer; Puritan swab.

[0292] Experimental procedure Nasal samples were collected using a Puritan swab and the nasal matrix was resuspended in 1.5ml of collection buffer. Next, the nasal samples suspended in 250 microliters of collection buffer were placed in an ultra-low temperature tube with two metal disks at its bottom and 10% Chelex 100. Inactivated SARS-COV2 virus at 32,000 copies per milliliter was added to the ultra-low temperature tube.

[0293] For the method of the present disclosure, the ultra-low temperature tube was then placed under a vibrating magnetic field generated by an induction coil and then heated under high pressure for 40, 60, 80, and 100 seconds, reaching 100°C, 103°C, 108°C, and 110°C respectively. For the control, exactly the same sample using the same percentage of Chelex in the same buffer was heated in a heating block at 98°C for 5 minutes.

[0294] Results Figure 1 shows the results of RT-PCR experiments according to the conditions described in the experimental procedure (high-pressure heating at 100 °C or above for 40, 60, 80, and 100 seconds) and the standard conditions of heating at 98 °C for 300 seconds. Each sample preparation method was performed on 10 μL samples and 20 μL samples to evaluate any effects from the sample volume. All the conditions tested efficiently neutralized PCR inhibitors from nasal specimens, as indicated by an average cycle threshold of 33 or less. Moreover, heating 10 μL samples at high pressure above 100 °C for 40, 60, and 80 seconds resulted in an average cycle threshold of 31 or less, equivalent to heating for 300 seconds at 98 °C. Further, for 20 μL samples, an average cycle threshold of less than 31 was achieved for all the tested conditions including high-pressure heating above 100 °C. Therefore, by adding more volume to the preparation, the sensitivity of PCR increased and there was no reactivation of PCR inhibitors in sample preparation.

[0295] The results shown in Figure 1 demonstrate that the method of the present disclosure gives superior results compared to the conventional method of heating at 95 - 98 °C. For example, 60 seconds of high-pressure heating reaching 103 °C is clearly superior to the standard conditions of 300 seconds at 98 °C, providing a lower average cycle threshold in one-fifth of the time. (Example 2) Effect of Temperature on Sample Preparation for RT-PCR Efficiency

[0296] In this example, the effect of temperature on sample preparation was tested. RT-PCR was performed to determine the efficiency of sample preparation at different temperatures (95 °C, 105 °C, 120 °C, and 130 °C).

[0297] Materials Used Glass ampoule; 10% Chelex 100; Collection buffer: 10 mM Tris / EDTA, 1 mM NaCl, 0.02% azide; 1 micromolar concentration of heat-stable single-stranded nucleic acid-binding protein; Heating block with aluminum beads; CDC Sars-CoV2 RT-PCR primers; QiaAmp viral RNA kit.

[0298] Experimental procedure The heating block was set to 95°C, 105°C, 120°C, and 130°C. 250 microliters of nasal matrix was resuspended in the collection buffer and 32000 copies of inactivated SARS-COV2 were added. This 250 microliter sample was then placed in a glass ampoule before being hermetically sealed. The tube was placed in the heating block set at the appropriate temperature for 2 minutes. The supernatant was collected and RT-PCR was performed in a commercially available thermocycler. For the control, an exact sample in the same buffer was also tested at the same temperature.

[0299] Results Figure 2 represents the mean cycle threshold for the detection of inactivated SARS-CoV-2 in (1) simple buffer solution (dark dots in the graph) and (2) swab samples. Each of these samples was subjected to different temperatures (95°C, 105°C, 120°C, and 130°C) for 2 minutes.

[0300] The results shown in Figure 2 demonstrate that the method of the present disclosure provides efficient neutralization of PCR inhibitors. As seen in Figure 2, the average cycle thresholds for the swab and buffer samples heated at 95°C are 36 and 30, respectively. The higher average cycle threshold for the swab sample (light gray dots, upper left) compared to the buffer sample (dark dots, lower left) indicates the PCR inhibitors remaining in the nasal sample after heating that are not present in the buffer solution. Therefore, the standard method of heating at 95°C was unable to inactivate all of the PCR inhibitors in the nasal swab sample after 2 minutes. In contrast, after autoclaving at 130°C for 2 minutes, both the nasal swab sample (light gray dots, right) and the buffer sample (dark dots, right) showed essentially the same average cycle threshold (average cycle threshold of about 30), indicating almost complete inactivation of all the PCR inhibitors present in the nasal swab sample.

[0301] Furthermore, the results presented in Figure 2 demonstrate that the method of the present disclosure does not cause detectable nucleic acid degradation. The average cycle threshold for the buffer sample was consistent across all temperatures examined (average cycle threshold of about 30), demonstrating that autoclaving the buffer sample at a high temperature of about 130°C did not cause significant nucleic acid degradation in the buffer sample. Moreover, since the nasal swab sample (light gray dots, right) showed the same average cycle threshold as the buffer sample (dark dots, right), no obvious degradation was identified. Considering Figure 2, it is proven that the method of the present disclosure achieves efficient inactivation of PCR inhibitors without degrading nucleic acids. (Example 3) The autoclaving method matches the industrial standard procedure with respect to the time required for PCR amplification

[0302] In this example, the method of the present disclosure was compared with a standard sample preparation method using the QiaAmp Viral RNA Kit, and the PCR amplification efficiency of each method was evaluated with different concentrations of analyte (SARS-CoV2 copies).

[0303] Materials Used Glass ampoule; 10% Chelex 100; Collection buffer: 10 mM Tris / EDTA, 1 mM NaCl, 0.02% azide; 1 micromolar concentration of heat-stable single-stranded nucleic acid-binding protein; Heating block with aluminum beads; CDC Sars-CoV2 RT-PCR primers; QiaAmp viral RNA kit.

[0304] Experimental procedure Several aliquots of 250 microliters of nasal matrix were resuspended in collection buffer, and to each aliquot, different concentrations of inactivated SARS-CoV2 (200, 500, 1000, 2000, 4000, 8000, 16000, and 32000 copies of inactivated SARS-CoV2 per milliliter) were added.

[0305] For the method of the present disclosure (FAST PCR Prep), the heating block was set at 130 degrees Celsius. Each 250 microliter sample was then placed in a glass ampoule before being airtight sealed. The tube was placed at 130 degrees Celsius for 2 minutes. The supernatant was collected and RT-PCR was performed in a commercially available thermocycler.

[0306] In parallel, as a control, a second copy of the sample aliquot having the same concentration of copies of SARS-CoV2 per milliliter was purified using the QiaAmp viral RNA kit according to the manufacturer's instructions. Each experimental group was performed in duplicate.

[0307] Results Figure 3 shows that the average cycle threshold from the method of the present disclosure (labeled "FASTPCR Superheating Prep") is similar to the average cycle threshold obtained from sample preparation using the Qiagen Viral RNA kit prep. Similar average cycle threshold values between the two methods were observed across multiple concentrations of analyte in nasal specimens, with higher analyte concentrations resulting in lower average cycle threshold values for each method. The results shown in Figure 3 thereby demonstrate that the PCR efficiency of the method of the present disclosure matches the performance of the Qiagen Viral RNA kit for the detection of SARS-COV2 in a significantly shorter time. (Example 4) High-pressure heating method in the absence of a chelating agent

[0308] In this example, different agents such as RNase inhibitors and / or reducing agents were tested in sample preparation for RT-PCR using the method of the present disclosure.

[0309] Materials Used Stainless steel cup; collection buffer containing 10 mM Tris / EDTA, 1 mM NaCl; induction heating system; and respiratory syncytial virus (RSV) RT-PCR primers.

[0310] Experimental Procedure A 250 μL sample of nasal swab matrix was collected and resuspended in the collection buffer. 32,000 copies of inactivated respiratory syncytial virus (RSV) were added to the collection and nasal swab matrix mixture. Prior to heating, aliquots of a portion of the sample were added with (a) none, (b) 1 mM DTT, (c) 200 U RNase (Ambion) alone, or (d) a combination of both 1 mM DTT and 200 U RNase. The 250 μL sample was then placed in a stainless steel container prior to airtight sealing. The container was then placed in an induction heating system set at 150 °C measured at the cup surface for 20 seconds. After the high-pressure heating step, the supernatant from each sample was collected and RT-PCR was performed in a commercially available thermocycler.

[0311] For the control, exactly the same samples were tested in clean buffer using the same conditions.

[0312] Results Figure 4 shows a table of the mean cycle thresholds for the detection of inactivated RSV RNA in both clean buffer and pooled swabs under four conditions: (1) no additive (first column); (2) 200 U of RNase inhibitor only (second column); (3) 1 mM of DTT only (third column); and (4) both 1 mM of DTT and 200 U of RNase inhibitor (fourth column). Chelating agents were not added to any of these test groups. UD indicates not detected, and N / A indicates not applicable.

[0313] The results indicate that sample preparation of pooled swab matrices using heat under pressure in the absence of Chelex is at least effective (mean cycle thresholds of 34.9 and 33.1, respectively) in the presence of an RNase inhibitor or a reducing agent (such as 1 mM DDT). The combination of a reducing agent and an RNase inhibitor in the collection buffer provides even better results for sample preparation of pooled swab matrices using the heat under pressure method. The mean cycles of RT-PCR from sample preparations using both a reducing agent and an RNase inhibitor were similar to those of control samples using clear buffer (mean cycle thresholds of 30.8 and 28.9, respectively), indicating almost complete inactivation of PCR inhibitors in the pooled swab matrix samples.

[0314] According to the results in Figure 4, the heat under pressure method for sample preparation provides effective neutralization of PCR inhibitors even in the absence of Chelex. This method is particularly effective in the presence of a reducing agent such as DDT, an RNase inhibitor, and / or a combination of a reducing agent and an RNase inhibitor. In addition, the results shown in Figure 4 indicate almost complete neutralization of PCR inhibitors when RNase is added in combination with a reducing agent.

[0315] Therefore, even in the absence of a chelating agent, the high-pressure heating method of the present disclosure is effective for sample preparation (e.g., inactivation of PCR inhibitors), especially in the presence of additives (e.g., reducing agents, RNAse inhibitors, and / or combinations thereof). (Example 5) High-pressure heating across multiple sample origins

[0316] In this example, it was shown that the method of the present disclosure provides similar PCR detection and sensitivity for various nucleic acid-containing samples compared to standard sample preparation methods using the Qiagen Viral RNA kit or the Qiagen DNeasy UltraClean Microbe DNA kit.

[0317] Materials used Metal heating container; induction coil; collection buffer (1×TE buffer and 2 mg / mL BSA); Chelex-100 Chelating Resin (final concentration in the treated sample before high-pressure heating: 12 wt / vol% of the treated sample); DTT (final concentration in the treated sample before high-pressure heating: 1 mM); thermostable Thermococcus kodakarensis (KOD) (final concentration in the treated sample before high-pressure heating: 0.5 μM); nucleic acid-containing sample; centrifuge; PCR primers targeting nucleic acids in the nucleic acid-containing sample; Qiagen Viral RNA kit; Qiagen DNeasy UltraClean Microbe DNA kit. The treated sample is a mixture of the nucleic acid-containing sample, collection buffer, Chelex-100 Chelating Resin, Thermococcus kodakarensis (KOD), and DTT.

[0318] Nucleic acid-containing sample The following nucleic acid-containing samples were tested in this example: SARS-CoV-2, HeLa mammalian cells, S. cerevisiae yeast, B. cereus bacterial spores, B. subtilis bacteria, and C. albicans fungi.

[0319] Experimental procedure The nucleic acid-containing sample was first prepared for PCR by the high-pressure heating method. First, the nucleic acid-containing samples were individually diluted using a collection buffer (1×TE buffer and 2 mg / mL BSA). The concentrations of the diluted nucleic acid samples in the collection buffer are shown in Figure 5. For each sample, a metal heating container was prepared using the following reagent components: Chelex-100 Chelating Resin (48 mg), DTT (3.2 μL), and Thermococcus kodakarensis (KOD) (8 μL), BSA (2 mg / mL). For each run, one of the diluted nucleic acid-containing samples (388.8 μL) was pipetted into a metal heating container containing Chelex-100 Chelating Resin, Thermococcus kodakarensis, and DTT. The metal heating container was sealed with an airtight cap, placed in an induction coil, and heated for 15 seconds. The metal heating container was cooled at ambient temperature until it could be safely handled. The metal heating container was placed in a centrifuge and spun to collect the material at the bottom of the container. The metal heating container was then opened, and the sample was aspirated into a microcentrifuge tube. The microcentrifuge tube was spun to pellet the Chelex-100 Chelating Resin, and the supernatant was collected for PCR analysis.

[0320] In parallel, control samples were also prepared from the original diluted nucleic acid sample aliquots. These samples were not subjected to high-pressure heating and were purified using a commercially available nucleic acid purification kit. For samples containing SARS-CoV-2, the QiaAmp Viral RNA kit was used according to the manufacturer's instructions. For samples containing HeLa mammalian cells, S. cerevisiae yeast, B. cereus bacterial spores, B. subtilis bacteria, and C. albicans fungi, the Qiagen DNeasy UltraClean Microbe DNA kit was used according to the manufacturer's instructions to purify the nucleic acids. The microbial DNA extraction protocol required 1.8 mL of sample and was performed in 70 minutes.

[0321] Samples prepared by high-pressure heating and control samples prepared by a commercially available purification kit were then used for PCR analysis. PCR amplification was performed on Applied Biosystems Quantstudio 3 and Quantstudio 5 PCR instruments. Each experimental group was performed in duplicate.

[0322] Results The results showed that the average cycle threshold from the method of the present disclosure (labeled "high-pressure heating") was similar to the average cycle threshold obtained from sample preparation using the Qiagen Viral RNA kit prep or the Qiagen DNeasy UltraClean Microbe DNA kit across different nucleic acid-containing samples. Figure 5 shows a table of the average cycle thresholds for the detection of inactivated nucleic acid samples from each sample origin. High-pressure heating for 15 seconds gave equivalent (within error range) or faster Ct values for each sample. The results shown in Figure 5 thereby demonstrate that the PCR efficiency of the method of the present disclosure matches the performance of commercial products for the detection of nucleic acids from multiple sample origins, including SARS-CoV-2, mammalian cells, yeast, bacteria, and fungi, in a significantly shorter time. (Example 6) Modification of the temperature gradient rate in nucleic acid sample preparation by high-pressure heating

[0323] In this example, different high-pressure heating vessels were created for high-pressure heating at different heating temperature gradient rates. The temperature was estimated using an internal temperature sensor inside the closed vessel and correlated with the external temperature infrared sensor that evaluated the external temperature of the closed vessel. Table 1 shows the different heating vessels created, as well as the heating method (high-pressure heating time) and heating temperature gradient rate for each vessel.

[0324] Table 1. High-pressure heating temperature gradient rate

Table 1-1

Table 1-2

[0325] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for analyzing nucleic acids, (a) A step of heating a sample containing nucleic acids in a closed heating chamber under high pressure conditions over a gradient time from a first temperature to a second temperature exceeding 105 degrees Celsius, thereby producing a heat-treated sample; and (b) Step of analyzing the nucleic acid from the heat-treated sample. Methods that include...

2. The method according to claim 1, wherein the gradient time is 3 to 50 seconds.

3. The method according to claim 1, wherein the step of heating the sample to be processed is performed with a temperature gradient of 5 degrees Celsius per second to 50 degrees Celsius per second.

4. The method according to claim 1, further comprising the steps of maintaining the sample to be treated at the second temperature for a maintenance time of 5 to 120 seconds after step (a) and before step (b), and cooling the sample to be heat-treated.

5. The processed sample includes a body sample containing the nucleic acid, i) The physical sample is selected from blood samples, tear samples, saliva samples, mucus samples, sputum samples, fecal samples, cerebrospinal fluid samples, and urine samples; ii) The nucleic acid has not been extracted, isolated, or otherwise purified from the body sample. The method according to claim 1.

6. The method according to claim 1, wherein the processed sample comprises non-lysed cells containing the nucleic acid, and the nucleic acid has not been extracted, isolated, or otherwise purified from the heat-treated sample prior to step (b).

7. The method according to claim 6, wherein the non-lysed cells are fungal cells.

8. The method according to claim 6, wherein the non-lysed cells are bacterial cells.

9. The method according to claim 1, wherein the nucleic acid has not been extracted, isolated, or otherwise purified from the heat-treated sample prior to step (b).

10. The method according to claim 1, wherein the processed sample comprises a plurality of molecular amplification inhibitors.

11. The method according to claim 10, wherein the plurality of molecular amplification inhibitors comprises (i) a plurality of reagents capable of binding to the nucleic acid, (ii) a plurality of DNases, (iii) a plurality of RNases, or (iv) a plurality of proteases.

12. The method according to claim 11, wherein the plurality of molecular amplification inhibitors comprises a plurality of RNases.

13. The method according to claim 10, wherein the heating step inactivates at least 60% of the plurality of molecular amplification inhibitors.

14. The method according to claim 1, wherein the processed sample comprises one or more reagents selected from a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, and a stabilizer.

15. The method according to claim 14, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent.

16. The method according to claim 1, wherein the nucleic acid is RNA.

17. The method according to claim 1, wherein the treated sample has a pH of about 8.0 to about 12.

0.

18. The method according to claim 1, wherein the heating step in (a) includes induction heating.

19. The method according to claim 1, wherein the analytical step of (b) includes a step of amplifying the nucleic acid by polymerase chain reaction (PCR) to thereby produce a PCR product.

20. The method according to claim 19, wherein the analytical step of (b) includes a step of detecting the PCR product, the PCR product being detectable after the nucleic acid has been amplified from 10 to 55 molecular amplification cycles.

21. The method according to claim 20, wherein the PCR product is detectable after the nucleic acid has been amplified from 28 to 35 molecular amplification cycles.

22. The method according to claim 1, wherein the step of analyzing in (b) comprises sequencing the nucleic acid or its amplification product.

23. The method according to claim 1, wherein the second temperature is 120 degrees Celsius to 160 degrees Celsius.

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