Sample preparation method

Abstract

Provided herein are methods for preparing a sample comprising nucleic acids. In some embodiments, the method includes heating a sample above its boiling point to rapidly extract nucleic acids from biological entities present in the sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 348,750, filed on June 3, 2022, the entire contents of which are incorporated herein by reference for all purposes. Background Art

[0003] During the sample preparation for nucleic acid analysis, two methods of extraction and purification of nucleic acid are mainly used. The first method includes purification of nucleic acid from other molecular species present in the sample by extraction. This method generally includes phenol / chloroform extraction and then ethanol precipitation, using a chaotropic lysing agent and then using an ion exchange resin, or using a silicone resin. These methods provide high-quality nucleic acid (for example, high-purity nucleic acid), but are time-consuming and involve many complicated steps. The second method is generally referred to as non-extraction, and the method uses physical, enzyme or chemical means to crack cells or microorganisms and amplify nucleic acids without the need to undergo further purification. The second method is simpler and usually faster than the first method, but the nucleic acid preparation quality provided is not as good as the first method.

[0004] In order to effectively amplify nucleic acid in the second method, it is necessary to neutralize the amplification inhibitor (e.g., PCR inhibitor) present in the sample so that molecular amplification of nucleic acid can occur before detection. In addition, nucleic acid must not be degraded so that it can be amplified and detected. For example, metal ions present in cells and / or tissues inhibit some amplification enzymes and may cause nucleic acid degradation. In addition, natural enzymes present in biological samples can inhibit molecular amplification (e.g., proteases) or degrade nucleic acids (e.g., nucleases). The current means for inactivating these amplification inhibitors (e.g., PCR inhibitors) rely on diluting the sample at least 10 times, which reduces the sensitivity of the entire assay. In addition, storing diluents in fluid boxes can cause the complexity of the entire manufacturing and shorten the shelf life of the care point device. Therefore, before nucleic acid amplification and detection, a simpler and faster effective sample processing method is highly desired. Summary of the invention

[0005] In one aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) heating a treated sample containing nucleic acids from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (b) analyzing nucleic acids from the heat-treated sample.

[0006] In some embodiments, treating the sample comprises one or more reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

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

[0008] In some embodiments, treating the sample comprises a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0009] In some embodiments, the first temperature is between 4 degrees Celsius and 40 degrees Celsius, and the second temperature is between 101 degrees Celsius and 160 degrees Celsius.

[0010] In some embodiments, the ramp time is from 3 to 50 seconds.

[0011] In some embodiments, heat treating the sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

[0012] In some embodiments, the method further comprises, after step (a) and before step (b), maintaining the treated sample at the second temperature for a holding time of 0 seconds to 120 seconds before a cooling time.

[0013] In some embodiments, the processed sample comprises a body sample containing nucleic acid, wherein: i) the body sample is selected from a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a stool sample, a cerebrospinal fluid sample, and a urine sample; and ii) the nucleic acid is not extracted, isolated, or otherwise purified from the body sample.

[0014] In another aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) providing a body sample selected from a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a stool sample, a cerebrospinal fluid sample and a urine sample; wherein the body sample contains nucleic acids; (b) heating a treated sample containing the body sample from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (c) analyzing the nucleic acids from the heat-treated sample; wherein the nucleic acids are not extracted, separated or otherwise purified from the body sample.

[0015] In some embodiments, the body sample is a saliva sample or a mucus sample.

[0016] In some embodiments, the body sample is collected from a human subject via a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0017] In some embodiments, the body sample comprises a pathogen or a portion of a pathogen.

[0018] In some embodiments, the pathogen or a portion of a pathogen is selected from a virus or a portion of a virus, a bacterium or a portion of a bacterium, a protozoa or a portion of a protozoa, a yeast or a portion of a yeast, and a fungus or a portion of a fungus.

[0019] In some embodiments, the pathogen is a virus or a portion of a virus.

[0020] In some embodiments, the virus is SARS-CoV2.

[0021] In some embodiments, the treated sample comprises unlysed cells containing nucleic acids, and wherein the nucleic acids are not extracted, isolated, or otherwise purified from the heat-treated sample prior to analyzing the nucleic acids from the heat-treated sample.

[0022] In another aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) heating a treated sample containing unlysed cells containing nucleic acids from a first temperature to a second temperature greater than 100 degrees Celsius in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample; wherein prior to step (b), the nucleic acids are not extracted, separated or otherwise purified from the heat-treated sample.

[0023] In some embodiments, the unlysed cells are selected from the group consisting of bacterial cells, fungal cells, and mammalian cells.

[0024] In some embodiments, treating the sample comprises one or more reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0025] In another aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) heating a treated sample containing nucleic acids from a first temperature to a second temperature greater than 100 degrees Celsius in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample; wherein the treated sample comprises one or more reagents selected from a chelator, a single-stranded nucleic acid binding protein, a reducing agent and a stabilizer.

[0026] In some embodiments, treating the sample comprises at least two agents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0027] In some embodiments, treating the sample comprises at least three reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0028] In some embodiments, treating the sample comprises a chelator, a single-stranded nucleic acid binding protein, and a reducing agent.

[0029] In some embodiments, treating the sample comprises a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0030] In some embodiments, the concentration of the chelating agent is 5% to 25% by weight to volume of the treated sample; the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM; the concentration of the reducing agent in the treated sample is 0.1 mM to 2 mM; or the concentration of the stabilizer in the treated sample is 500 ng / mL to 10 mg / mL.

[0031] In some embodiments, the chelating agent is an insoluble chelating agent.

[0032] In some embodiments, the insoluble chelating agent comprises styrene divinyl benzene copolymer.

[0033] In some embodiments, the chelating agent is a soluble chelating agent.

[0034] In some embodiments, the soluble chelating agent comprises EDTA.

[0035] In some embodiments, the stabilizer is bovine serum albumin or gelatin.

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

[0037] In some embodiments, the nucleic acid is RNA.

[0038] In some embodiments, the nucleic acid is selected from the group consisting of a viral nucleic acid, a bacterial nucleic acid, a protozoan nucleic acid, a eukaryotic nucleic acid, and a fungal nucleic acid.

[0039] In some embodiments, the nucleic acid is a viral nucleic acid.

[0040] In some embodiments, the processed sample contains 50 copies / mL to 10 9 Copies / mL of nucleic acid.

[0041] In some embodiments, the volume of the processed sample is between 100 μL and 5 mL.

[0042] In some embodiments, the pH of the treated sample is from about 8.0 to about 12.0.

[0043] In some embodiments, the treated sample further comprises a protease.

[0044] In some embodiments, treating the sample further comprises a nuclease inhibitor.

[0045] In some embodiments, processing the sample is heated with a heat source.

[0046] In some embodiments, the heat source comprises an induction heater, a heating element, or microwaves.

[0047] In some embodiments, the heat source comprises an induction heater.

[0048] In some embodiments, the enclosed heating chamber includes a thermally conductive material.

[0049] In some embodiments, the enclosed heating chamber includes a ferromagnetic material.

[0050] In some embodiments, the nucleic acid is not substantially degraded after heating.

[0051] In some embodiments, the method further comprises amplifying the nucleic acid by polymerase chain reaction (PCR) after heating, thereby generating a PCR product.

[0052] In some embodiments, the method further comprises detecting the PCR product.

[0053] In some embodiments, PCR products are detectable after fewer cycles of molecular amplification than would be required without heating the processed sample.

[0054] In some embodiments, PCR products are detectable after fewer cycles of molecular amplification than would be required if the sample were heat treated at a temperature below 100 degrees Celsius.

[0055] In some embodiments, PCR products are detectable after amplifying the nucleic acid using 10 to 55 cycles of molecular amplification.

[0056] In some embodiments, PCR products are detectable after 28 to 35 cycles of molecular amplification.

[0057] In some embodiments, analyzing the nucleic acid includes detecting the nucleic acid, sequencing the nucleic acid, or genotyping the nucleic acid.

[0058] In some embodiments, analyzing the nucleic acid comprises detecting the nucleic acid by fluorescence detection.

[0059] In some embodiments, heat treating the sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 20 degrees Celsius per second.

[0060] In another aspect, the present disclosure provides a method for inactivating molecular amplification inhibitors in a treated sample containing nucleic acids, the method comprising: under high pressure heating conditions, heating the treated sample containing i) nucleic acid and ii) multiple molecular amplification inhibitors from a first temperature to a second temperature above 100 degrees Celsius within a ramp time in a closed heating chamber, thereby inactivating the molecular amplification inhibitors in the multiple amplification inhibitors and producing a heat-treated sample, wherein the nucleic acid is substantially not degraded.

[0061] In some embodiments, the first temperature is between 4 degrees Celsius and 40 degrees Celsius, and the second temperature is between 101 degrees Celsius and 160 degrees Celsius.

[0062] In some embodiments, the method further comprises maintaining the treated sample at the second temperature for a holding time prior to the cooling time.

[0063] In some embodiments, the hold time is from 0 seconds to 120 seconds.

[0064] In some embodiments, the method inactivates at least 70% of the plurality of molecular amplification inhibitors.

[0065] In some embodiments, the method inactivates at least 90% of the plurality of molecular amplification inhibitors.

[0066] In some embodiments, the processed sample comprises a body sample containing nucleic acid, wherein: i) the body sample is selected from a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a stool sample, a cerebrospinal fluid sample, and a urine sample; and ii) the nucleic acid is not extracted, isolated, or otherwise purified from the body sample.

[0067] In some embodiments, the bodily sample is a saliva sample.

[0068] In some embodiments, the bodily sample is a mucus sample.

[0069] In some embodiments, the body sample is collected from an animal or human subject.

[0070] In some embodiments, the body sample is collected from an animal or human subject via a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0071] In some embodiments, the processed sample comprises unlysed cells containing nucleic acid.

[0072] In some embodiments, treating the sample comprises one or more reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0073] In some embodiments, treating the sample comprises at least two agents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0074] In some embodiments, treating the sample comprises at least three reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0075] In some embodiments, treating the sample comprises a chelator, a single-stranded nucleic acid binding protein, and a reducing agent.

[0076] In some embodiments, treating the sample comprises a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

[0077] In some embodiments, the concentration of the chelating agent is 5% to 25% by weight to volume of the treated sample; the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM; the concentration of the reducing agent in the treated sample is 0.1 mM to 2 mM; or the concentration of the stabilizer in the treated sample is 500 ng / mL to 10 mg / mL.

[0078] In some embodiments, the pH of the treated sample is from about 8.0 to about 12.0.

[0079] In some embodiments, the treated sample further comprises a protease.

[0080] In some embodiments, treating the sample further comprises a nuclease inhibitor.

[0081] In some embodiments, heat treating the sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

[0082] In another aspect, the present disclosure provides a method for analyzing nucleic acids, the method comprising: (a) heating a treated sample containing nucleic acids from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (b) analyzing the nucleic acids from the heat-treated sample; wherein the heating occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

[0083] Incorporation by reference

[0084] All publications, patents, and patent applications mentioned in this specification are herein 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The novel features of the invention are set forth with particularity in the appended claims.A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and the accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized.

[0086] Figure 1 Results of RT-PCR experiments following a method of the present disclosure (using high temperature for 40 seconds, 60 seconds, 80 seconds, or 100 seconds) and a traditional sample preparation method (heating to 98 degrees Celsius for 300 seconds) are shown. The results show that high PCR efficiency (indicated by low average cycle threshold) is achieved in a shorter time (e.g., 60 seconds) than with the traditional method (i.e., 300 seconds).

[0087] Figure 2It was shown that treating nasal swab samples containing inactivated SARS-CoV-2 virus with the methods of the present disclosure (e.g., autoclaving to 130 degrees Celsius) resulted in increased PCR efficiency, as represented by lower mean cycle threshold values.

[0088] Figure 3 It was shown that sample preparation using the method of the present disclosure provided similar PCR detection and sensitivity compared to sample preparation using the Qiagen Viral RNA kit.

[0089] Figure 4 It is shown that in addition to or as an alternative to chelators, the methods of the present disclosure can utilize various additives including reducing agents, RNase inhibitors, or combinations thereof.

[0090] Figure 5 It was shown that sample preparation using the methods of the present disclosure provided similar PCR detection and sensitivity for a variety of nucleic acid-containing samples compared to sample preparation using the Qiagen Viral RNA kit or the Qiagen DNeasy UltraCleanMicrobe DNA kit.

[0091] Figure 6A-6C Several views of a high pressure heating vessel are shown. Fig. 6A shows a side view of a high pressure heating vessel, Figure 6B A top view of a high pressure heating vessel is shown, and Figure 6C A bottom view of the high pressure heating vessel is shown.

[0092] Figure 7A-7B A high pressure heating vessel is shown where the sample inlet is a duckbill valve. Fig. 7A A side view of the high pressure heating vessel and duckbill valve is shown, and Figure 7B A cross-sectional view of the high pressure heating vessel and duckbill valve is shown.

[0093] Figure 8 A photo of the induction heating platform is provided.

[0094] Fig. 9 An enclosed autoclave chamber is shown including an outlet passage housed within an autoclave vessel.

[0095] Fig.10 A photograph is provided of a bottom view of the disclosed high pressure heating vessel showing a laser valve sealing the disclosed outlet passage.

[0096] Figure 11A-11B The movement of the sample during sample injection and sample release and a magnified view of the laser valve are shown. Fig.11AMovement of the sample during sample injection is shown, and a magnified view of the outlet channel and closed laser valve is provided. Fig. 11B The movement of the sample during sample release is shown, and a magnified view of the outlet channel and sample movement through the open laser valve is provided. DETAILED DESCRIPTION

[0097] Provided herein is a method for preparing nucleic acid samples for rapid release of high-quality nucleic acid from microorganisms or eukaryotic cells using a temperature higher than 100 degrees Celsius in a form ready for downstream applications. In short, the sample (e.g., body sample) collected from the object is heated (e.g., high pressure heating) for a short period of time (e.g., less than 60 seconds) at a temperature higher than 100 degrees Celsius (e.g., 130 degrees Celsius) to crack the cells and / or viruses in the sample, releasing the nucleic acid (both DNA and RNA) present therein. The method described in the present disclosure not only provides an effective technique for releasing nucleic acid from other components (e.g., proteins or other cellular components) in biological samples, but also these methods can also destroy the amplification inhibitors (e.g., PCR inhibitors) present in biological samples. Therefore, the method described in the present disclosure provides a nucleic acid sample that can be used for downstream applications (such as, for example, molecular amplification, nucleic acid detection, nucleic acid sequencing, etc.), without further diluting the sample. In some cases, the method described in the present disclosure provides a nucleic acid sample that can be used for molecular amplification and / or PCR analysis, without the need to improve the concentration of DNA polymerase, reverse transcriptase or RNA enzyme inhibitors. In addition, when using an ultrafast thermal cycler, the method is as effective as more cumbersome nucleic acid extraction methods to achieve high sensitivity of PCR in less than 10 minutes of thermal cycling. Therefore, the method of the present disclosure can save time and resources (such as using purification kits and expensive enzymes) in sample preparation steps.

[0098] Although it is known that some nucleic acids (such as, for example, RNA) can be degraded at high temperatures, the present disclosure provides surprising results, i.e. nucleic acids (comprising both DNA and RNA) can be effectively amplified after being heated to a temperature higher than 100 degrees Celsius. This discovery is particularly surprising in terms of RNA that 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 (for example, a sealed container) that can maintain a high vapor pressure, and the sample is heated to a temperature higher than 100 degrees Celsius (for example, high pressure heating) while the sample is in the device or container. In some embodiments, before the heating step, the container is sealed to prevent steam from escaping during the high pressure heating step, and promote the pressure accumulation in the container. In some embodiments, the pressure in the container accumulates to, for example, about 15 to 40PSI (including atmospheric pressure). In addition, the sealed container can prevent the sample solution (for example, an aqueous sample solution) from boiling, so that the sample solution reaches a temperature far higher than 100 degrees Celsius (for example, 130 degrees Celsius). Preventing the sample solution from boiling can result in cell and / or viral lysis and neutralization of enzyme inhibitors present in the sample without damaging nucleic acids (DNA or RNA).

[0099] In addition, in some aspects of the present disclosure, additives are added to the sample (e.g., before heating, during heating, or after heating). For example, a chelating agent (such as Chelex) can be added to the sample before high pressure 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, a chelating agent is not added to the sample (e.g., when a reducing agent and / or an RNase inhibitor is added to the sample, a chelating agent can be omitted in sample preparation). The chelating agent can exist during heating, or can be used before the heating step and removed before heating, or can be omitted altogether. Even at the high temperature used in the method of the present disclosure, such a resin will chelate metal ions, otherwise the metal ions will interfere with nucleic acid amplification and / or detection. In some embodiments, the pH is acidic (pH is 5). In some other embodiments, the pH is close to neutral. In some embodiments, the pH of the sample is alkaline (e.g., pH is 8 or higher), which helps to neutralize RNA nuclease activity at the temperature used in the present disclosure. Surprisingly, the present disclosure provides a sample preparation method that does not cause significant degradation of nucleic acids (even RNA) even under alkaline conditions and high temperatures. In some embodiments, a thermostable single-stranded nucleic acid binding protein (SSB) is added to the sample mixture prior to heating to further protect the nucleic acids (e.g., RNA) during the heating step, even if the pH is neutralized to a pH at which the nucleic acids are active.

[0100] RNA nucleases can sometimes survive harsh heating conditions. Therefore, in some embodiments, additives (such as reducing agents (e.g., DTT or TCEP), with or without chelating agents) are added using the methods of the present disclosure to completely neutralize RNA nucleases.

[0101] The present disclosure provides a sample preparation method for providing high-quality nucleic acid sample preparation, and the nucleic acid sample prepared by the method can be easily used for downstream applications (such as molecular amplification and detection). Compared with conventional sample processing methods, the whole process can use less time and a shorter workflow, thereby providing a more effective nucleic acid sample preparation method.

[0102] definition

[0103] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. 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 is not necessarily to be construed as representing a substantial difference from what is generally understood in the art.

[0104] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the present disclosure. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the scope, this applies.

[0105] As used in the specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, including mixtures thereof.

[0106] The terms "determine," "measure," "assess," "evaluate," "measure," and "analyze" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element (element) is present (e.g., detecting). These terms may include quantitative determination, qualitative determination, or both quantitative and qualitative determination. Evaluations may be relative or absolute. In addition to determining whether something is present or not, depending on the context, "detecting the presence of..." may also include determining the amount of its presence.

[0107] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" can be a biological entity containing genetic material. A 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 derived from a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal, such as a human. A subject may be diagnosed or suspected of being at high risk for a disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk for a disease.

[0108] As used herein, the term "about" means plus or minus 10% of the number. The term "about" a range means the range minus 10% of its lower limit and plus 10% of its upper limit.

[0109] As used herein, the term "molecular amplification" refers to a measure or method or test for detecting nucleic acid in a sample. This can be used for example for experimental research, clinical medicine development, infectious disease diagnosis, gene cloning and industrial quality control. Molecular amplification can also be used as a part of a diagnostic test. The term "molecular amplification" referred to herein is intended to encompass all methods designed to amplify (e.g., replicate, copy, etc.) nucleic acids to generate more nucleic acid copies in a sample. Molecular amplification includes nucleic acid amplification, enzyme amplification (e.g., PCR), isothermal amplification and / or other alternative amplification methods developed in the art.

[0110] 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. PCR methods 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").

[0111] As used herein, the term "biological sample" means a sample containing nucleic acid / biological agents, such as clinical samples (e.g., cell components, mucosa, nasal swabs, whole blood, plasma, serum, urine, tissues, cells, etc.), agricultural samples, environmental samples (e.g., soil, earth, minerals, water, air), food samples, forensic samples, or any other biological samples. The sample may contain infectious agents, for example, such as viral, bacterial or parasitic infectious agents. About "whole blood", it means blood containing white blood cells and red blood cells, platelets, plasma, and any infectious agents that may be present, for example, collected by venous sampling. Clinical samples may be from humans or animal sources. The sample analyzed may be solid or liquid in nature. Obviously, when using solid materials, these solid materials are first dissolved in a suitable solution known in the art.

[0112] As used herein, the term "chelating agent" refers to a reagent for removing metal ions in a solution. Chelating agents include soluble chelating agents (e.g., reagents that can form water-soluble complexes with metal ions), and the soluble chelating agents include, for example, sodium tripolyphosphate, EDTA, DTPA, NTA, citrate, etc. Chelating agents also include insoluble chelating agents (e.g., reagents that form insoluble complexes with metal ions), and the insoluble chelating agents include, for example, sodium triphosphate, zeolite A, etc. In some embodiments, chelating agents are used to remove metal ion impurities or inactivate and / or inhibit molecular amplification inhibitors in samples, thereby improving the molecular amplification efficiency of samples. Chelating agents can be used to improve the quality of nucleic acid samples before performing molecular amplification.

[0113] As used herein, the term "reducing agent" refers to a reagent that provides electrons to an electron acceptor. In some embodiments, the term "reducing agent" refers to a reagent for reducing the disulfide combined in a protein. Reducing agents include, for example, 2-mercaptoethanol, 2-mercaptoethylamine-HCl, TCEP, cysteine-HCl, dithiothreitol (DTT), TCEP-HCl, thiol-based reducing agents, guanidine-HCl and urea, etc. As described in the present disclosure, other reducing agents known in the art can be used.

[0114] As used herein, the term "hyperbaric" or "hyperbaric" refers to a condition where the pressure is greater than or higher than the normal pressure at the same altitude level.

[0115] As used herein, the term "nucleic acid" refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), a DNA-RNA hybrid, and analogs of DNA or RNA produced 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 nucleosome small molecules. It can be RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), heteronuclear RNA (hnRNA), or small hairpin RNA (shRNA).

[0116] Sample preparation method

[0117] The present disclosure provides a sample preparation method for applying 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 sample preparation method for nucleic acid in a body sample, which does not involve extracting, separating, or otherwise purifying nucleic acid from a body sample. The present disclosure provides a method for inactivating a molecular amplification inhibitor (e.g., an RNase present in saliva or mucus) in a sample, thereby preparing the sample for further processing.

[0118] The present disclosure also provides a method for analyzing nucleic acids in biological samples, the method comprising preparing a sample using the method disclosed herein, and subsequently analyzing the nucleic acids. The sample preparation method described herein can prepare a sample for molecular amplification of nucleic acids 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 degree of nucleic acid degradation and improve the detectability of nucleic acids or their molecular amplification products for analysis.

[0119] Analyzing nucleic acids can be performed using a variety of techniques known in the art, such as sequencing nucleic acids (e.g., sequencing by synthesis, sequencing by hybridization, nanopore sequencing, etc.), genotyping amino acids (e.g., genotyping by hybridization, genotyping by sequencing, etc.), or detecting nucleic acids (e.g., detection by hybridization, antibody binding, fluorescence, radioisotope detection, etc.). For example, in some embodiments, detecting nucleic acids comprises hybridizing the nucleic acid to a fluorescently labeled nucleic acid comprising a sequence complementary to at least a portion of the nucleic acid using methods known in the art.

[0120] In some embodiments, analyzing nucleic acid comprises one or more of the following: detecting nucleic acid, sequencing nucleic acid, and genotyping nucleic acid. In some embodiments, analyzing nucleic acid comprises detecting nucleic acid. In some embodiments, analyzing nucleic acid comprises sequencing nucleic acid. In some embodiments, analyzing nucleic acid comprises genotyping nucleic acid.

[0121] In some embodiments, analyzing nucleic acid includes analyzing the molecular amplification product of nucleic acid (e.g., a DNA copy of RNA produced during PCR amplification of RNA in a sample using a reverse transcriptase). For example, in some embodiments, detecting nucleic acid includes detecting the molecular amplification product of nucleic acid. In some embodiments, sequencing nucleic acid includes sequencing the molecular amplification product of nucleic acid. In some embodiments, genotyping nucleic acid includes genotyping the molecular amplification product of nucleic acid. It will be appreciated by those of ordinary skill in the art that many means for analyzing nucleic acid are known in the art, all of which are compatible with the methods disclosed herein and are contemplated herein.

[0122] In some embodiments, molecular amplification includes enzyme 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), autonomous sequence replication (3SR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HAD), branch amplification method (RAM), transcription-mediated determination (TMA), nickase amplification reaction (NEAR), recombinase polymerase amplification (RPA) or whole genome amplification (WGA).

[0123] In some embodiments, molecular amplification includes polymerase chain reaction ("PCR"). In some embodiments, PCR includes reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), quantitative real-time PCR (qPCR), digital PCR (dPCR), digital droplet PCR (ddPCR), microfluidics PCR, multiplex PCR, variable number tandem repeat sequence (VNTR) PCR, asymmetric PCR, nested PCR, quantitative PCR, hot start PCR, touchdown PCR, assembly PCR, colony PCR, suicide PCR, low denaturation temperature co-amplification-PCR (COLD-PCR), cDNA end rapid amplification (RACE) PCR, two-tailed PCR, ligation-mediated PCR, methylation-specific PCR (MSP), sequence-specific PCR (or ISSR-PCR), RNase H-dependent PCR (rhPCR) or Vectorette PCR.

[0124] In some embodiments, nucleic acid can be analyzed after molecular amplification (e.g., PCR) of nucleic acid. In some embodiments, nucleic acid is analyzed as a part of downstream applications. In some embodiments, downstream applications include molecular amplification or sequencing. In some embodiments, downstream applications include probe hybridization and / or detection probes.

[0125] In some aspects, the method of the present disclosure includes heat treatment sample. " Treatment sample " referred to herein is a sample (for example, aqueous solution or suspension) containing at least nucleic acid and optional reagent (such as enzyme or chelating agent). In certain embodiments, the treatment sample can be a nucleic acid biological sample collected from an object and diluted with water or buffer and optionally comprising one or more reagents. The biological sample can be a body sample, such as saliva or mucus. The biological sample can include cells (for example, unlysed cells). In certain embodiments, before dilution and / or addition of reagent, the nucleic acid sample collected from the object is separated or purified, for example, the nucleic acid sample is separated or purified from the cell, protein or other biological product in the biological sample. In certain embodiments, before dilution and / or addition of reagent, the nucleic acid sample collected from the object is not separated or purified, for example, the nucleic acid sample is not separated or purified from the cell, protein or other biological product in the biological sample.

[0126] In some aspects, the method includes heating the treated sample to a temperature higher than 100 degrees Celsius in a closed heating chamber under high pressure heating conditions. For example, a closed heating chamber can substantially prevent air and steam from entering or leaving the chamber. In some cases, the air flow rate in and out of the closed heating chamber is negligible. The closed heating chamber can remain closed during high pressure heating. In some embodiments, "high pressure heating conditions" referred to herein are conditions for heating to generate higher pressures in a closed heating chamber than outside a closed heating chamber. In some embodiments, the temperature referred to herein is each the average temperature of the duration of a sample heating step or a part thereof. In some embodiments, the temperature referred to herein is the temperature in a closed container. In some embodiments, the temperature referred to herein is the temperature of a heating device for high pressure heating samples. In some cases, an internal temperature sensor in a closed container is used to estimate the temperature. In some cases, an external temperature infrared sensor is used to associate the internal temperature by evaluating the external temperature of a closed container. In some aspects, the heating of the treated sample occurs at a temperature ramp rate of, for example, 6 degrees Celsius per second to 20 degrees Celsius per second. In some aspects, the methods of the present disclosure include heating the treated sample from a first temperature to a second temperature greater than 100 degrees within a ramp time.

[0127] In some aspects, the methods of the present disclosure include analyzing nucleic acids in a body sample selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In one aspect, the method includes providing a body sample, and heating a processed sample comprising the body sample. In some embodiments, the nucleic acids are not extracted, separated, or otherwise purified from the body sample.

[0128] In some aspects, the method of the present disclosure includes heating a treated sample comprising unlysed cells containing nucleic acids, thereby producing a heat-treated sample. In some embodiments, the method also includes analyzing nucleic acids. In some embodiments, before analyzing nucleic acids, nucleic acids are not extracted, separated or otherwise purified from the heat-treated sample.

[0129] In some aspects, the treated sample comprising nucleic acids further comprises one or more agents selected from the group consisting of a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent. In some embodiments, the treated sample is a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent.

[0130] In some aspects, the present disclosure provides a method for inactivating a molecular amplification inhibitor in a treated sample containing nucleic acids. In some aspects, the method includes heating a treated sample containing i) nucleic acid and ii) multiple molecular amplification inhibitors. In one aspect, the method includes heating the treated sample to a temperature higher than 100 degrees Celsius in a closed heating chamber under high pressure heating conditions, thereby inactivating the molecular amplification inhibitors in the multiple amplification inhibitors and producing a heat-treated sample. In another aspect, the method includes heating the treated sample from a first temperature to a second temperature higher than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure heating conditions, thereby inactivating the molecular amplification inhibitors in the multiple amplification inhibitors and producing a heat-treated sample, wherein the nucleic acid is substantially not degraded. In some embodiments, the method also includes detecting nucleic acids.

[0131] In some aspects, the methods described herein include heating the treated sample to a temperature of more than 100 degrees Celsius in a closed heating chamber under high pressure. In some embodiments, the method includes heating the treated sample under high pressure to a temperature of more than 100 degrees Celsius. The temperature may be higher than the boiling point of water. In some embodiments, the treated sample does not boil under high pressure at a temperature of more than 100 degrees. In some embodiments, the method includes heating the treated sample under high pressure to a temperature of 100 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 101 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 105 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 110 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 120 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 130 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 140 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 150 degrees Celsius to 160 degrees Celsius. In some embodiments, the temperature is 100 degrees Celsius to 140 degrees Celsius. In some embodiments, the temperature is 110 degrees Celsius to 140 degrees Celsius. In some embodiments, the temperature is 120 degrees Celsius to 140 degrees Celsius. In some embodiments, the temperature is 130 degrees Celsius to 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.

[0132] In some aspects, the heating of processing sample occurs and reaches the first time period.In some embodiments, the first time 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 time 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 time 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 time 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 time 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 time 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.

[0133] In some embodiments, the first time 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 time 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 time period is, for example, 1 minute to 5 minutes, 2 minutes to 5 minutes, 3 minutes to 5 minutes, 4 minutes to 5 minutes.

[0134] In some aspects, the method further comprises, after the treatment sample is heated to a temperature higher than 100 degrees Celsius: before the cooling time, the treatment sample is kept at this temperature for a holding time. In some embodiments, the holding time is 0 seconds to 300 seconds, 0 seconds to 250 seconds, 0 seconds to 200 seconds, 0 seconds to 150 seconds, 0 seconds to 120 seconds, 0 seconds to 100 seconds, 0 seconds to 50 seconds, 0 seconds to 40 seconds, 0 seconds to 30 seconds, 0 seconds to 20 seconds or 1 second to 10 seconds. In some embodiments, the holding time is 5 seconds to 300 seconds, 5 seconds to 250 seconds, 5 seconds to 200 seconds, 5 seconds to 150 seconds, 5 seconds to 100 seconds, 5 seconds to 50 seconds, 5 seconds to 40 seconds, 5 seconds to 30 seconds, 5 seconds to 20 seconds or 5 seconds to 10 seconds.

[0135] During the hold time, the closed heating chamber can remain closed (eg, substantially preventing air and steam from entering or leaving the chamber).

[0136] 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.

[0137] In some embodiments, the cooling time is 10 seconds to 120 seconds. In some embodiments, the cooling time is 30 seconds to 120 seconds. In some embodiments, the cooling time is 60 seconds to 120 seconds. In some embodiments, the cooling time is 90 seconds to 120 seconds. During the cooling time, the closed heating chamber can remain closed (e.g., substantially preventing air and steam from entering or leaving the chamber). In some embodiments, the nucleic acid is substantially not degraded after heating.

[0138] In some aspects, heating the treated sample comprising the nucleic acid occurs at a temperature ramp rate. In some embodiments, the temperature ramp rate is calculated as the time derivative of the temperature. In some embodiments, the temperature ramp rate is calculated as the average rate of change of temperature relative to time. In some embodiments, the temperature ramp 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 21 degrees Celsius per second, at least 22 degrees Celsius per second, at least 23 degrees Celsius per second, at least 24 degrees Celsius per second, at least 25 degrees Celsius per second, at least 26 degrees Celsius per second, at least 27 degrees Celsius per second, at least 28 degrees Celsius per second, at least 29 degrees Celsius per second, at least 30 degrees Celsius per second, at least 31 degrees Celsius per second, at least 32 degrees Celsius per second, at least 33 degrees Celsius per second, at least 34 degrees Celsius per second, at least 35 degrees Celsius per second, at least 36 degrees Celsius per second, at least 37 degrees Cel 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.

[0139] In some embodiments, the temperature ramp rate is 0.5 degrees Celsius per second to 50 degrees Celsius per second, 0.5 degrees Celsius per second to 40 degrees Celsius per second, 0.5 degrees Celsius per second to 35 degrees Celsius per second, 0.5 degrees Celsius per second to 30 degrees Celsius per second, 0.5 degrees Celsius per second to 25 degrees Celsius per second, 0.5 degrees Celsius per second to 20 degrees Celsius per second, or 0.5 degrees Celsius per second to 15 degrees Celsius per second.

[0140] In some embodiments, the temperature ramp rate is 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.

[0141] In some embodiments, the temperature ramp rate is 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. In some embodiments, the nucleic acid is not substantially degraded after heating.

[0142] In some aspects, the method described herein includes heating the treated sample comprising the nucleic acid from a first temperature to a second temperature higher than 100 degrees Celsius in a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat treated sample. During heating from the first temperature to the second temperature, the closed heating chamber can remain closed (e.g., substantially preventing air from entering or leaving the chamber). In some embodiments, the treated sample does not boil under high pressure conditions at a second temperature higher than 100 degrees. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 100 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 101 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 105 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 110 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 120 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 130 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 140 degrees Celsius to 160 degrees Celsius.

[0143] In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 100 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 101 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 105 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 110 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 120 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 20 degrees Celsius to 30 degrees Celsius, and the second temperature is 130 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is between 20 degrees Celsius and 30 degrees Celsius, and the second temperature is between 140 degrees Celsius and 160 degrees Celsius.

[0144] In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 100 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 101 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 105 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 110 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 120 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 130 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 140 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is at least 150 degrees Celsius. In some embodiments, the first temperature is between 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 160 degrees Celsius.

[0145] In some embodiments, the ramp 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 ramp 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.

[0146] In some embodiments, the method for analyzing nucleic acid further comprises after (a): keeping the treated sample at a second temperature for a holding time before the cooling time. In some embodiments, the holding time is 0 seconds to 300 seconds. In some embodiments, the holding time is 5 seconds to 300 seconds. In some embodiments, the holding time is 30 seconds to 300 seconds. In some embodiments, the holding time is 60 seconds to 300 seconds. In some embodiments, the holding time is 90 seconds to 300 seconds. In some embodiments, the holding time is 120 seconds to 300 seconds. In some embodiments, the holding time is 150 seconds to 300 seconds. In some embodiments, the holding time is 180 seconds to 300 seconds. In some embodiments, the holding time is 240 seconds to 300 seconds. In some embodiments, the holding time is 270 seconds to 300 seconds.

[0147] In some embodiments, the holding time is 0 seconds to 120 seconds. In some embodiments, the holding time is 10 seconds to 120 seconds. In some embodiments, the holding time is 30 seconds to 120 seconds. In some embodiments, the holding time is 60 seconds to 120 seconds. In some embodiments, the holding time is 90 seconds to 120 seconds. During the holding time, the closed heating chamber can remain closed (e.g., substantially preventing air and steam from entering or leaving the chamber).

[0148] In some embodiments, the cooling time is 0 seconds to 300 seconds. In some embodiments, the cooling time is 5 seconds to 300 seconds. In some embodiments, the cooling time is 30 seconds to 300 seconds. In some embodiments, the cooling time is 60 seconds to 300 seconds. In some embodiments, the cooling time is 90 seconds to 300 seconds. In some embodiments, the cooling time is 120 seconds to 300 seconds. In some embodiments, the cooling time is 150 seconds to 300 seconds. In some embodiments, the cooling time is 180 seconds to 300 seconds. In some embodiments, the cooling time is 240 seconds to 300 seconds. In some embodiments, the cooling time is 270 seconds to 300 seconds.

[0149] In some embodiments, the cooling time is 10 seconds to 120 seconds. In some embodiments, the cooling time is 30 seconds to 120 seconds. In some embodiments, the cooling time is 60 seconds to 120 seconds. In some embodiments, the cooling time is 90 seconds to 120 seconds. During the cooling time, the closed heating chamber can remain closed (e.g., substantially preventing air and steam from entering or leaving the chamber).

[0150] In some aspects, heat treating a sample in a closed chamber generates a pressure within the chamber. In some embodiments, the pressure within the chamber is, for example, 1 to 200 PSI, 10 to 200 PSI, 20 to 200 PSI, 30 to 200 PSI, 40 to 200 PSI, 50 to 200 PSI, 10 to 100 PSI, 20 to 100 PSI, 30 to 100 PSI, 40 to 100 PSI, 50 to 100 PSI, 60 to 100 PSI, 70 to 100 PSI, 80 to 100 PSI, or 90 to 100 PSI, each of which exceeds 1 atm. In some embodiments, the pressure in the chamber is, for example, 10 to 100PSI, 10 to 90PSI, 10 to 80PSI, 10 to 70PSI, 10 to 60PSI, 10 to 50PSI, 10 to 40PSI, 10 to 30PSI, 10 to 20PSI, 20 to 100PSI, 20 to 90PSI, 20 to 80PSI, 20 to 70PSI, 20 to 60PSI, 20 to 50PSI, 20 to 40PSI, 20 to 30PSI, 30 to 100PSI, 30 to 90PSI, 30 to 80PSI, 30 to 70PSI, 30 to 60PSI, 30 to 50PSI or 30 to 40PSI, each of which exceeds 1atm. In some embodiments, the pressure in the chamber is 40 to 60PSI exceeding 1atm. In some embodiments, the pressure within the chamber is 40 to 50 PSI in excess of 1 atm. In some embodiments, the nucleic acid is not substantially degraded after heating.

[0151] Any means for heating the sample can be used, including but not limited to contact heating in a heat block, induction heating, microwave heating, nanophoton heating, or any other heating means known in the art. In some aspects, the sample is heated with a heat source. In some embodiments, the heat source comprises an induction heater, a heating element, or a microwave. For example, in some embodiments, a heat block or a hot bath can be used, such as by filling with preheated aluminum beads. In some embodiments, the heating of the biological sample is high pressure heating. In some embodiments, the sample is heated with a heat source at high pressure.

[0152] For example, in some embodiments, the sample is heat treated using a heat block or a heat bath that is preheated at a temperature ranging from 100 degrees Celsius to 300 degrees Celsius, or more preferably 100 degrees Celsius to 150 degrees Celsius, wherein the autoclave is placed on the heat block or the heat bath and left for, e.g., 1 second to 10 minutes after reaching 100 degrees Celsius. In another embodiment, the container is autoclaved for 20 to 40 seconds after reaching a temperature of 100 degrees Celsius or more.

[0153] 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 the paramagnetic material can be placed in direct contact with the sample in a heating container made of a non-paramagnetic heat-resistant material (such as a plastic polymer). This provides an extreme temperature ramp rate that can bring the sample above 100 degrees Celsius in less than 30 seconds, which allows sample preparation to be completed in a minute or less.

[0154] In some embodiments, the laser can be used to heat the processed sample in the container at a temperature higher than 100 degrees Celsius (e.g., high pressure heating). Nanoparticles can be added to the heating system to capture the laser energy and convert it directly into the sample. This technology provides a very high heating ramp rate compatible with overheated sample preparation. In some embodiments, the heat source is a laser. In some embodiments, nanoparticles are added to the laser.

[0155] In some aspects, the disclosure provides methods for heating a processed sample comprising 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 cell or a 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, protozoan nucleic acid, eukaryotic nucleic acid and fungal nucleic acid. In some embodiments, the nucleic acid is a viral nucleic acid.

[0156] In some embodiments, a processed sample comprising a body sample containing nucleic acid is heated in a closed heating chamber under high pressure. In some embodiments, the body sample comprises a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymph, bile, synovial fluid, cyst fluid, ascites, pleural fluid, eye fluid, interstitial fluid, cervical fluid and urine. In some embodiments, the body sample is selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid and urine. In some embodiments, the body sample comprises mucus. In some embodiments, the body sample comprises a body fluid sample, tissue or cell of a subject. In some embodiments, nucleic acid is not extracted, separated or otherwise purified from the body sample.

[0157] In some aspects, a body sample can be collected from an object (e.g., a human being). The body sample can be collected from the object by a nasopharyngeal swab, a cervical swab, or a nasal swab. In some cases, the body sample can include a pathogen or a portion of a pathogen. In some embodiments, the pathogen or a portion of a pathogen is selected from a virus or a portion of a virus, a bacterium or a portion of a bacterium, a protozoan or a portion of a protozoan, a yeast or a portion of a yeast, and a fungus or a portion of a fungus. In some embodiments, the pathogen is a blood-borne pathogen or a portion of a blood-borne pathogen. In some embodiments, the pathogen is a respiratory pathogen or a portion of a respiratory pathogen.

[0158] In some embodiments, a treated sample comprising a body sample containing nucleic acids is heated to a temperature greater than 100 degrees Celsius in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample. In some aspects, the nucleic acids are analyzed after high pressure heating. In some cases, the nucleic acids are not extracted, isolated, or otherwise purified from the body sample. For example, in some cases, the nucleic acids are not extracted by phenol chloroform extraction, or are not purified by a commercial nucleic acid purification kit, or are not purified by column chromatography.

[0159] In some aspects, under high pressure conditions, a processed sample containing cells containing nucleic acids is heated in a closed heating chamber. The cell can be an unlysed cell. In some embodiments, the cell is embedded in a biomatrix (such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine or saliva). In some embodiments, the cell is embedded in nasal mucus. In some embodiments, the cell is a bacterial cell, such as Bacillus subtilis (B.subtilis), Escherichia coli (E.Coli) or Streptococcus pyogenes (S.Pyogenes). In some embodiments, the cell is a fungal cell, such as Candida albicans (C.albicans). In some embodiments, the cell is a yeast cell, such as Saccharomyces cerevisiae (S.cerevisiae). In some embodiments, the cell is a mammalian cell, such as Chinese hamster ovary cell, BHK cell or mouse C127 cell. In some embodiments, the cell is a human cell, such as HeLa cell. In some embodiments, the processed sample comprises bacterial spores, such as Bacillus cereus (B.cereus) bacterial spores.

[0160] In some embodiments, a treated sample comprising unlysed cells containing nucleic acids is heated to a temperature greater than 100 degrees Celsius in a closed heating chamber 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 are not extracted, separated, or otherwise purified from the heat-treated sample prior to analyzing the nucleic acids.

[0161] 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 can be a reagent that binds to nucleic acids. The molecular amplification inhibitor can be a reagent that degrades nucleic acids. In some cases, the molecular amplification inhibitor can be a nuclease. In some cases, the molecular amplification inhibitor is a DNA enzyme. In other cases, the molecular amplification inhibitor is an RNA enzyme.

[0162] In some aspects, the method includes heating a treated sample comprising i) nucleic acid and ii) a plurality of molecular amplification inhibitors. In one aspect, the method includes heating the treated sample to a temperature higher than 100 degrees Celsius in a closed heating chamber under high pressure heating conditions, thereby inactivating the molecular amplification inhibitors in a plurality of amplification inhibitors and producing a heat-treated sample. In another aspect, the method includes heating the treated sample from a first temperature to a second temperature higher than 100 degrees Celsius in a ramp time in a closed heating chamber under high pressure heating conditions, thereby inactivating the molecular amplification inhibitors in the plurality of amplification inhibitors and producing a heat-treated sample, wherein the nucleic acid is substantially undegraded. In some embodiments, the degraded nucleic acid does not exceed, for example, 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some embodiments, the method also includes amplifying nucleic acids using molecular amplification. In some embodiments, the method also includes detecting nucleic acids.

[0163] In some aspects, the method inactivates at least one molecular amplification inhibitor in the treated sample. In some embodiments, the method inactivates at least 60% of a plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 70% of a plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 80% of a plurality of molecular amplification inhibitors. In some embodiments, the method inactivates at least 90% of a plurality of molecular amplification inhibitors.

[0164] In some aspects, the nucleic acid is not substantially degraded after heating (such as high pressure heating). In some embodiments, for example, the nucleic acid degraded after high pressure heating is no more than 20%, no more than 15%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than 1%. In some embodiments, for example, after high pressure heating, the nucleic acid is 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% complete.

[0165] In some aspects, nucleic acid is analyzed after high pressure heating. In some aspects, the method for analyzing nucleic acid includes amplifying nucleic acid by molecular amplification (for example, by polymerase) using any method disclosed elsewhere herein after high pressure heating. Molecular amplification can occur during real-time polymerase chain reaction (real-time PCR), transcription-mediated amplification (TMA) or loop-mediated isothermal amplification (LAMP). The molecular amplification of nucleic acid can produce molecular amplification products. In some embodiments, the method for analyzing nucleic acid also includes amplifying the nucleic acid by polymerase chain reaction (PCR) after heating, thereby producing PCR products. In some embodiments, the method also includes detecting PCR products. In some cases, PCR products are detected by fluorescent signals emitted during amplification.

[0166] In some embodiments, the molecular amplification product (e.g., PCR product) is detectable after fewer molecular amplification cycles than would be required without high pressure heating of the processed sample. In some embodiments, the molecular amplification product (e.g., PCR product) is detectable after fewer molecular amplification cycles than would be required to heat the processed sample at a temperature below 100 degrees Celsius. In some embodiments, the nucleic acid is detectable after fewer molecular amplification cycles than would be required to heat the sample for the same amount of time at a temperature below the boiling point of the sample.

[0167] In some embodiments, the molecular amplification products (e.g., PCR products) are detectable after 10 to 50 cycles of molecular amplification, 15 to 50 cycles of molecular amplification, 20 to 50 cycles of molecular amplification, 25 to 50 cycles of molecular amplification, 26 to 50 cycles of molecular amplification, 27 to 50 cycles of molecular amplification, 28 to 50 cycles of molecular amplification, 29 to 50 cycles of molecular amplification, or 30 to 50 cycles of molecular amplification.

[0168] In some embodiments, the molecular amplification products (e.g., PCR products) are detectable after 10 to 40 cycles of molecular amplification, 20 to 40 cycles of molecular amplification, 25 to 40 cycles of molecular amplification, 26 to 40 cycles of molecular amplification, 27 to 40 cycles of molecular amplification, 28 to 40 cycles of molecular amplification, 29 to 40 cycles of molecular amplification, or 30 to 40 cycles of molecular amplification.

[0169] In some embodiments, the molecular amplification product (e.g., PCR product) is detectable after 10 to 35 molecular amplification cycles, 20 to 35 molecular amplification cycles, 25 to 35 molecular amplification cycles, 26 to 35 molecular amplification cycles, 27 to 35 molecular amplification cycles, 28 to 35 molecular amplification cycles, 29 to 35 molecular amplification cycles, or 30 to 35 molecular amplification cycles. In some embodiments, the molecular amplification product (e.g., PCR product) is detectable after 28 to 35 molecular amplification cycles. In some embodiments, the nucleic acid is detectable after, for example, 25 to 35 molecular amplification cycles, 25 to 34 molecular amplification cycles, 25 to 33 molecular amplification cycles, 25 to 32 molecular amplification cycles, 25 to 31 molecular amplification cycles, 25 to 30 molecular amplification cycles, 25 to 29 molecular amplification cycles, or 25 to 28 molecular amplification cycles.

[0170] In some embodiments, the molecular amplification product (e.g., PCR product) is detectable after 25 or more molecular amplification cycles. In some embodiments, the nucleic acid is detectable after, for example, 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.

[0171] In some embodiments, molecular amplification products (e.g., PCR products) are detected after amplifying nucleic acids using 10 to 40 cycles of molecular amplification, 10 to 35 cycles of molecular amplification, 20 to 35 cycles of molecular amplification, 25 to 35 cycles of molecular amplification, 26 to 35 cycles of molecular amplification, 27 to 35 cycles of molecular amplification, 28 to 35 cycles of molecular amplification, 29 to 35 cycles of molecular amplification, or 30 to 35 cycles of molecular amplification.

[0172] In some embodiments, the detection of nucleic acid occurs simultaneously with the molecular amplification of nucleic acid. In some embodiments, the detection of nucleic acid and the molecular amplification of nucleic acid occur sequentially. In some embodiments, the detection of nucleic acid occurs after each molecular amplification cycle.

[0173] In some embodiments, the method for analyzing nucleic acid also includes at least one of the following: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acid also includes at least two of the following: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acid also includes: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acid includes detecting nucleic acid by fluorescence detection.

[0174] As described herein, the heated processed sample contains at least nucleic acid and optional reagent (such as enzyme or chelating agent). In some embodiments, nucleic acid is DNA. In some embodiments, nucleic acid is RNA. In some embodiments, nucleic acid derives from eukaryotic cells or prokaryotic cells. In some embodiments, nucleic acid derives from viruses. In some embodiments, nucleic acid is selected from viral nucleic acid, bacterial nucleic acid, protozoan nucleic acid, eukaryotic nucleic acid and fungal nucleic acid. In some embodiments, nucleic acid is viral nucleic acid.

[0175] In some embodiments, the nucleic acid is extracted from an organism selected from a prokaryotic organism. In some embodiments, the nucleic acid is extracted from an organism selected from a eukaryotic organism. In some embodiments, the nucleic acid is extracted from a parasite. In some embodiments, the nucleic acid is extracted from a virus, a bacterium, a fungus, an animal, or a plant.

[0176] 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.

[0177] 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.

[0178] In some cases, the nucleic acid is in a body sample. In some embodiments, the nucleic acid is not extracted, separated or otherwise purified from a body sample. The body sample can be selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid and urine. In some embodiments, the body sample includes a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymph fluid, bile, synovial fluid, cyst fluid, ascites, pleural fluid, eye fluid, interstitial fluid, cervical fluid and urine. In some embodiments, the body sample includes mucus. In some embodiments, the body sample includes a body fluid sample, tissue or cell of the subject.

[0179] In some aspects, a body sample can 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 a marmoset, a macaque, a chimpanzee), a rodent (such as a mouse, a rat, a gerbil, a puffer pig, a hamster, a cotton rat, a naked mole rat), a rabbit, a livestock mammal (such as a goat, a sheep, a pig, a cow, a horse, a camel), a pet animal (such as a dog, a cat), or a zoo mammal. In some embodiments, the subject is a human.

[0180] The body sample can be collected from the subject by a nasopharyngeal swab, a cervical swab, or a nasal swab. In some cases, the body sample may include a pathogen or a portion of a pathogen. In some embodiments, the pathogen or a portion of a pathogen is selected from a virus or a portion of a virus, a bacterium or a portion of a bacterium, a protozoa or a portion of a protozoa, a yeast or a portion of a yeast, and a fungus or a portion of a fungus. In some embodiments, the pathogen is a blood-borne pathogen or a portion of a blood-borne pathogen. In some embodiments, the pathogen is a respiratory pathogen or a portion of a respiratory pathogen. In some embodiments, the respiratory pathogen includes a bacterial pathogen 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 portion of a virus. 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), human parainfluenza virus type I (HPIV-1), human parainfluenza virus type II (HPIV-2), human parainfluenza virus type III (HPIV-3), human parainfluenza virus type IV (HPIV-4), 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.

[0181] 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.

[0182] In some aspects, the treated sample comprises a bodily sample and is heated to a temperature greater than 100 degrees Celsius under high pressure conditions in a closed heating chamber, thereby producing a thermally treated sample.

[0183] In some aspects, the processed sample comprises a cell containing nucleic acid. The cell can be an unlysed cell. The cell can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a bacterial cell, such as Bacillus subtilis, Escherichia coli or Streptococcus pyogenes. In some embodiments, the cell is a fungal cell, such as Candida albicans. In some embodiments, the cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the cell is a mammalian cell, such as a Chinese hamster ovary cell, a BHK cell or a mouse C127 cell. In some embodiments, the cell is a human cell, such as a HeLa cell. In some embodiments, the cell is a bacterial spore, such as a Bacillus cereus bacterial spore.

[0184] In some embodiments, the cell can be in a body sample. In some embodiments, the cell is embedded in a biological matrix (such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine or saliva). In some embodiments, the cell is embedded in nasal mucus.

[0185] The treated sample can be an aqueous solution or a suspension. In some embodiments, the treated sample comprises an isolated nucleic acid diluted in a collection buffer. In other embodiments, the treated sample comprises a body sample (e.g., saliva or mucus) comprising nucleic acids diluted in a collection buffer. In further embodiments, the treated sample comprises an undiluted body sample containing nucleic acids. In some embodiments, the treated 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 mixture of low buffer capacity buffers. Examples of such buffers can be based on a Tris HCl buffer ranging from 1 mM to 50 mM, with or without EDTA in a concentration range of 0.5 mM to 1 mM.

[0186] In some embodiments, the pH of the treated sample is about 8.0 to about 12.0. In some embodiments, the pH of the treated sample is about 9.0 to about 12.0. In some embodiments, the pH of the treated sample is about 10.0 to about 12.0. In some embodiments, the pH of the treated sample is about 11.0 to about 12.0. In some embodiments, the pH of the treated sample is about 8.0 to about 11.0. In some embodiments, the pH of the treated sample is about 9.0 to about 11.0. In some embodiments, the pH of the treated sample is about 10.0 to about 11.0. In some embodiments, the pH of the treated sample is about 8.0 to about 10.0. In some embodiments, the pH of the treated sample is about 9.0 to about 10.0.

[0187] In some embodiments, the pH of the treated sample is higher than 7.0. In some embodiments, the pH of the treated sample is higher than 8.0. In some embodiments, the pH of the treated sample is higher than 8.5. In some embodiments, the pH of the treated sample is higher than 9.0. In some embodiments, the pH of the treated sample is higher than 9.5. In some embodiments, the pH of the treated sample is higher than 10.0. In some embodiments, the pH of the treated sample is higher than 10.5. In some embodiments, the pH of the treated sample is higher than 11.0.

[0188] In some embodiments, the pH of the treated sample is about 7.0. In some embodiments, the pH of the treated sample is about 8.0. In some embodiments, the pH of the treated sample is about 9.0. In some embodiments, the pH of the treated sample is about 10.0. In some embodiments, the pH of the treated sample is about 11.0. In some embodiments, the pH of the treated sample is about 12.0.

[0189] In some embodiments, the pH of the treated sample is about 8.0 to 11.0. In some embodiments, the pH of the treated sample is about 9.0 to 11.0. In some embodiments, the pH of the treated sample is about 10.0 to 11.0.

[0190] In some embodiments, the pH of the treated sample is about 8.0 to 12.0. In some embodiments, the pH of the treated sample is about 9.0 to 12.0. In some embodiments, the pH of the treated sample is about 10.0 to 12.0. In some embodiments, the pH of the treated sample is about 11.0 to 12.0.

[0191] In some embodiments, the pH of the treated sample is about 4.0 to about 7.0. In some embodiments, the pH of the treated sample is about 5.0 to about 7.0. In some embodiments, the pH of the treated sample is about 6.0 to about 7.0. In some embodiments, the pH of the treated sample is about 4.0 to about 6.0. In some embodiments, the pH of the treated sample is about 5.0 to about 6.0. In some embodiments, the pH of the treated sample is about 4.0 to about 5.0.

[0192] In some embodiments, the pH of the treated sample is about 3.0. In some embodiments, the pH of the treated sample is about 4.0. In some embodiments, the pH of the treated sample is about 5.0. In some embodiments, the pH of the treated sample is about 6.0. In some embodiments, the pH of the treated sample is about 7.0.

[0193] In some embodiments, the pH of the treated sample is less than about 3.0. In some embodiments, the pH of the treated sample is less than about 4.0. In some embodiments, the pH of the treated sample is less than about 5.0. In some embodiments, the pH of the treated sample is less than about 6.0. In some embodiments, the pH of the treated sample is less than about 7.0.

[0194] In some embodiments, the volume of the processed sample is the total volume of the mixture of the biological sample, the collection buffer and one or more additives before high pressure heating. In some embodiments, the volume of the processed sample is 100 μL to 5 mL. In some embodiments, the volume of the processed sample is 200 μL to 5 mL. In some embodiments, the volume of the processed sample is 300 μL to 5 mL. In some embodiments, the volume of the processed sample is 400 μL to 5 mL. In some embodiments, the volume of the processed sample is 500 μL to 5 mL. In some embodiments, the volume of the processed sample is 1 mL to 5 mL. In some embodiments, the volume of the processed sample is 1.5 mL to 5 mL. In some embodiments, the volume of the processed sample is 2 mL to 5 mL. In some embodiments, the volume of the processed sample is 2.5 mL to 5 mL. In some embodiments, the volume of the processed sample is 3 mL to 5 mL. In some embodiments, the volume of the processed sample is 3.5 mL to 5 mL. In some embodiments, the volume of the processed sample is 4 mL to 5 mL. In some embodiments, the volume of the processed sample is 4.5 mL to 5 mL.

[0195] In some embodiments, the processed sample contains 10 copies / mL to 10 9 In some embodiments, the processed sample contains 50 copies / mL to 10 9In some embodiments, the processed sample contains 100 copies / mL to 10 9 In some embodiments, the processed sample contains 10 3 Copies / mL to 10 9 In some embodiments, the processed sample contains 10 4 Copies / mL to 10 9 In some embodiments, the processed sample contains 10 5 Copies / mL to 10 9 In some embodiments, the processed sample contains 10 6 Copies / mL to 10 9 Copies / mL of nucleic acid.

[0196] In some embodiments, the processed sample contains 10 copies / mL to 10 8 In some embodiments, the processed sample contains 50 copies / mL to 10 8 In some embodiments, the processed sample contains 100 copies / mL to 10 8 In some embodiments, the processed sample contains 10 3 Copies / mL to 10 8 In some embodiments, the processed sample contains 10 4 Copies / mL to 10 8 In some embodiments, the processed sample contains 10 5 Copies / mL to 10 8 In some embodiments, the processed sample contains 10 6 Copies / mL to 10 8 Copies / mL of nucleic acid.

[0197] In some embodiments, the processed sample contains 10 copies / mL to 10 7 In some embodiments, the processed sample contains 50 copies / mL to 10 7 In some embodiments, the processed sample contains 100 copies / mL to 10 7 In some embodiments, the processed sample contains 10 3 Copies / mL to 10 7 In some embodiments, the processed sample contains 10 4 Copies / mL to 10 7In some embodiments, the processed sample contains 10 5 Copies / mL to 10 7 In some embodiments, the processed sample contains 10 6 Copies / mL to 10 7 Copies / mL of nucleic acid.

[0198] In some aspects, the processing sample comprises 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.

[0199] In some embodiments, the treated sample comprises a chelating agent. In some embodiments, the chelating agent is an insoluble chelating agent. In some embodiments, the insoluble chelating agent comprises a chelating resin. The chelating resin can be a polymer or a copolymer. The chelating resin can be a cation binding agent or a metal ion binding agent. The chelating resin can be in the form of microbeads. In some embodiments, the chelating agent comprises cross-linked polystyrene. The chelating agent can include one or more functional groups. One or more functional groups can include sulfonic acid or sulfonic acid groups; quaternary amino groups (e.g., trimethylammonium); primary, secondary and / or tertiary amino groups (e.g., polyvinylamine); or carboxylic acid or carboxylic acid groups. In some embodiments, the insoluble chelating agent comprises a styrene divinylbenzene copolymer. In some embodiments, the insoluble chelating agent comprises a Chelex resin or Chelex. In some embodiments, the Chelex is stored in a collection buffer.

[0200] 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, dimercaptopropanol, porphine, heme, hemoglobin or chlorophyll. In some embodiments, the chelating agent includes simple organic acids, such as oxalic acid, malic acid, rhodomycin or citric acid.

[0201] In some embodiments, the chelator is added prior to heating at a final concentration defined as a weight-to-volume percentage of the weight of the chelator to the volume of the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelator, a single-stranded nucleic acid binding protein, and a reducing agent). In some embodiments, the final concentration of the chelator in the treated sample prior to heating is 1% to 40%, 2.5% to 35%, 5% to 25%, 7.5% to 20%, or 10% to 15%, based on the weight-to-volume ratio of the weight of the chelator to the volume of the treated sample. In some embodiments, the final concentration of the chelator in the treated sample prior to heating is about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, or about 20%, based on the weight-to-volume ratio of the weight of the chelator to the volume of the treated sample.

[0202] In some embodiments, the processed 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 degrees Celsius to 170 degrees Celsius, 4 degrees Celsius to 160 degrees Celsius, 4 degrees Celsius to 150 degrees Celsius, 4 degrees Celsius to 140 degrees Celsius, 4 degrees Celsius to 130 degrees Celsius, or 4 degrees Celsius to 120 degrees Celsius. In some embodiments, the SSB protein is thermostable at a temperature of 90 to 170 degrees Celsius, 90 to 160 degrees Celsius, 90 to 150 degrees Celsius, 90 to 140 degrees Celsius, 90 to 130 degrees Celsius, 90 to 120 degrees Celsius, or 90 to 110 degrees Celsius.

[0203] In some embodiments, the final molar concentration of the SSB protein in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, and a reducing agent) prior to heating is 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.

[0204] In some embodiments, the final molar concentration of the SSB protein in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, and a reducing agent) prior to heating 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, about 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.

[0205] 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 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 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 neoapollo (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 Thermotoga thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid binding protein is selected from ETSSB, E. coli SSB, KOD SSB, TthSSB, TneSSB, TmaSSB, and TaqSSB.

[0206] In some embodiments, the treated sample comprises a reducing agent. In some embodiments, a reducing agent is added to the treated sample before heating. The reducing agent can be 2-mercaptoethanol, 2-mercaptoethylamine-HCl, TCEP, cysteine-HCl, dithiothreitol (DTT), TCEP-HCl, a thiol-based reducing agent, guanidine-HCl or urea.

[0207] In some embodiments, the final molar concentration of the reducing agent in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, and a reducing agent) prior to heating is 0.1 mM to 10 mM, 0.1 mM to 9 mM, 0.1 mM to 8 mM, 0.1 mM to 7 mM, 0.1 mM to 6 mM, 0.1 mM to 5 mM, 0.1 mM to 4 mM, 0.1 mM to 3 mM, 0.1 mM to 2 mM, 0.2 mM to 1.8 mM, 0.4 mM to 1.6 mM, 0.6 mM to 1.4 mM, or 0.8 mM to 1.2 mM. In some embodiments, the final molar concentration of the reducing agent in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, and a reducing agent) prior to heating 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.

[0208] In some embodiments, the treated sample comprises one or more reagents selected from chelators, single-stranded nucleic acid binding proteins, and reducing agents. In some embodiments, the treated sample comprises at least two reagents selected from chelators, single-stranded nucleic acid binding proteins, and reducing agents. In some embodiments, the treated sample comprises a chelator, a single-stranded nucleic acid binding protein, and a reducing agent. In some embodiments, the concentration of the chelator is 2.5% to 35% by weight to the volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 5 μM, and the concentration of the reducing agent in the treated sample is 0.1 mM to 5 mM. In some embodiments, the concentration of the chelator is 5% to 25% by weight to the volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM, and the concentration of the reducing agent in the treated sample is 0.1 mM to 2 mM. In some embodiments, the concentration of the chelator is 10% to 15% by weight to volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.3 μM to 0.7 μM, and the concentration of the reducing agent in the treated sample is 0.6 mM to 1.4 mM.

[0209] In some embodiments, the treated sample comprises a stabilizer. In some embodiments, the treated sample is mixed with a stabilizer before heating. In some cases, a stabilizer can help prevent nucleic acid degradation. In some cases, a stabilizer can help inactivate molecular amplification inhibitors during high pressure heating. In some embodiments, a stabilizer can stabilize proteins. In some embodiments, a stabilizer can stabilize nucleic acids. In some embodiments, a stabilizer can stabilize nucleic acids and proteins. A stabilizer can increase the viscosity of the treated sample. A stabilizer can help prevent or reduce aggregation between one or more proteins. A stabilizer can increase the solubility of a protein. A stabilizer can reduce non-specific binding between one or more components (e.g., proteins). A stabilizer can be a blocking agent. A stabilizer can be a component that stabilizes one or more components in the treated sample. In some embodiments, a stabilizer can stabilize nucleic acids. In some embodiments, a stabilizer can stabilize a chelating agent. In some embodiments, a stabilizer can stabilize a single-stranded nucleic acid binding protein. For example, a stabilizer can be bovine serum albumin (BSA). A stabilizer can be gelatin. In some embodiments, the concentration of the stabilizer in the treated sample is 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 concentration of the stabilizer in the treated sample is 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 concentration of the stabilizer in the treated sample is 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.

[0210] In some embodiments, the treated sample comprises one or more reagents selected from chelators, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the treated sample comprises at least two reagents selected from chelators, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the treated sample comprises at least three reagents selected from chelators, single-stranded nucleic acid binding proteins, reducing agents, and stabilizers. In some embodiments, the treated sample comprises chelators, single-stranded nucleic acid binding proteins, and reducing agents. In some embodiments, the treated sample comprises chelators, single-stranded nucleic acid binding proteins, and reducing agents. In some embodiments, the concentration of the chelator is 2.5% to 35% by weight to the volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 5 μM, the concentration of the reducing agent in the treated 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 chelator is 5% to 25% by weight to the volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM, the concentration of the reducing agent in the treated 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 chelator is 10% to 15% by weight to the volume of the treated sample, the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.3 μM to 0.7 μM, the concentration of the reducing agent in the treated sample is 0.6 mM to 1.4 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL.

[0211] In some embodiments, the treated sample comprises a protease. In some embodiments, the treated sample is mixed with a protease before heating. In some embodiments, the protease is, for example, a proteinase K. In some embodiments, the biological sample is mixed with a reducing agent and a protease before heating. In some embodiments, the final concentration of the protease in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, a reducing agent, and a protease) before heating is 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 the protease in the treated sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, a SSB protein, a reducing agent, and a protease) prior to heating 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.

[0212] In some embodiments, the treated sample comprises a nuclease inhibitor. In some embodiments, the treated sample comprises an RNase inhibitor. In some embodiments, the amount of the RNase inhibitor added before heating is, for example, about 1U, about 10U, about 50U, about 100U, about 150U, about 200U, about 250U, about 300U, about 350U, about 400U, about 450U, or about 500U.

[0213] In some embodiments, the amount of RNase inhibitor in the treated sample is about 1U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 10U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 50U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 100U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 150U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 200U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 250U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 300U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 350U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 400U to about 500U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 450 U to about 500 U.

[0214] In some embodiments, the amount of RNase inhibitor in the treated sample is about 1U to about 200U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 10U to about 200U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 50U to about 200U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 100U to about 200U. In some embodiments, the amount of RNase inhibitor in the treated sample is about 150U to about 200U.

[0215] In some embodiments, the treated sample further comprises a molecular amplification inhibitor. In some cases, the molecular amplification inhibitor can be a reagent that binds to nucleic acid. The molecular amplification inhibitor can be a reagent that degrades nucleic acid. In some cases, the molecular amplification inhibitor can be a nuclease. In some cases, the molecular amplification inhibitor is a DNA enzyme. In other cases, the molecular amplification inhibitor is an RNA enzyme. In some embodiments, the treated sample comprises one or more molecular amplification inhibitors that can be inactivated after heating.

[0216] As described herein, the sample can be heat treated in a closed heating chamber. The closed heating chamber can substantially prevent air and steam from entering or leaving the chamber. In some cases, the air flow in and out of the closed heating chamber is negligible. The closed heating chamber can remain closed during high pressure heating. In some embodiments, the closed heating chamber is a chamber in a heating container. The heating container and / or the closed heating chamber can be made of different materials. In some embodiments, the heating container and / or the closed heating chamber include glass. In some embodiments, the heating container and / or the closed heating chamber include high temperature polycarbonate. In some embodiments, the heating container and / or the closed heating chamber include thermally conductive materials. In some embodiments, the heating container and / or the closed heating chamber include metals. In some embodiments, the heating container and / or the closed heating chamber can 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 container and / or the closed heating chamber include conductive materials. In some embodiments, the heating container and / or the enclosed heating chamber comprises a ferromagnetic material, such as iron, nickel, cobalt. For example, the heating container and / or the enclosed heating chamber can be or comprise a glass ampoule, a plastic container, or a metal container. In some embodiments, the heating container and / or the enclosed heating chamber comprises an inductive susceptor 103 (e.g., a metal cup) ( FIGS. 6 , 7 , Fig. 9 ), the sensing receptor 103 can be used to communicate with the sensing platform 801 ( Figure 8 ) contact for induction heating to heat the sample.

[0217] In some embodiments, the heating vessel includes a heating chamber 702 (FIG. 7) that holds the processed sample during high pressure heating. The material of the vessel and / or heating chamber can be selected so that the heating chamber can have a temperature ramp 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 materials of the container and / or heating chamber can be selected so that the heating chamber can have a temperature ramp 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.

[0218] In some embodiments, the heating chamber is closed during high pressure heating and prevents air and steam from entering or leaving the chamber. In some embodiments, the closed heating chamber remains closed under high pressure conditions, wherein the pressure in the chamber is higher than the pressure outside the chamber. The material of the container or the material of the heating chamber can be selected so that the closed heating chamber can remain closed during high pressure heating (e.g., from room temperature to 160 degrees Celsius) or when the pressure in the heating chamber is 1 to 200 PSI exceeding 1atm.

[0219] In some embodiments, the closed heating chamber can be a glass ampoule or include a glass ampoule. The glass ampoule can include a molten sample inlet. In other embodiments, the closed heating chamber can be a plastic container or include a plastic container. The plastic container can include a plastic resin or a heat-resistant plastic including polycarbonate, high-density polypropylene, PEEK or PEI. For example, the plastic container can be a part of a cryogenic tube or a microfluidic box. In further embodiments, the closed heating chamber can be a metal container or include a metal container. The metal container can include an inductive receptor (e.g., a metal cup).

[0220] In some embodiments, the sealed heating chamber is produced by the heating chamber opened by sealing. The sealing operation may include the sample inlet opened by melting. The sealing operation may include the use of a cover, a lid, a stopper or a valve. The mode of the sealed heating chamber may be selected so that the sealed heating chamber can remain closed during high pressure heating.

[0221] In some embodiments, the heating container includes a sample inlet 101, and the sample can enter the heating chamber 102 through the sample inlet 101, as shown in FIG. 6, FIG. 7, Fig. 9 11. The sample inlet can be sealed to create a closed heating chamber within the heating container. In some embodiments, a cover, lid, plug, or valve is used to seal the sample inlet. In some embodiments, the sample inlet can include a valve. The valve can be a one-way valve 201 (e.g., a duckbill valve) ( Fig. 7A , Figure 7B ), the one-way valve 201 allows the sample to enter the heating chamber and prevents air and / or the sample from leaving the heating chamber. In some cases, the sample passes through the one-way valve 201 ( Fig. 7A , Figure 7B ) is introduced. The sample can be introduced manually using a pipette or injection needle. In other cases, the sample can be introduced by an automated system. The heating container can be part of a fluid system, and the one-way valve can be in contact with an upstream component of the fluid system. In some embodiments, the one-way valve can further prevent air and steam from leaving the heating chamber. The one-way valve provides a seal 202 (Figure 7) for the closed chamber during high pressure heating.

[0222] In some embodiments, the closed chamber is opened at a certain point in time to release the sample from the heating container. The closed chamber can be opened after high pressure heating. In some embodiments, a mechanical force is used to open the heating chamber. In some embodiments, the heating chamber is opened by removing a cover, a lid, a plug or a valve from the outlet. In some embodiments, the heating chamber is opened by opening the outlet valve 302 at the outlet. For example, the outlet valve 302 can be a laser valve that is initially closed and can be opened by shining a laser beam on the valve ( Fig.10 , Figure 11). In some cases, the outlet can be the same as the sample inlet or located in the same position as the sample inlet. In other cases, the outlet is located in a different position from the sample inlet.

[0223] In some embodiments, the sample can be released from the outlet. The outlet can be part of the outlet channel 301 in the heating container ( Fig. 9 , Fig.10 The outlet channel 301 may include an outlet valve 302 or may be in contact with the outlet valve 302 ( Fig. 9 , Fig.10 ). For example, in Fig.11A During sample injection in operation 1101 and during high pressure heating, the outlet channel 301 may initially be sealed by the outlet valve 302. The outlet channel 301 may then be unsealed by opening the outlet valve 302 for use, for example, Fig. 11B When the outlet valve 302 is opened, the sample can leave the heating chamber through the outlet channel 301. A force (e.g., centrifugal force) can be used to push the sample out of the heating chamber through the outlet channel 301. In some embodiments, the sample is released into the collection chamber 303 ( Fig. 9 ). In some embodiments, the collection chamber is part of the heating container. In some embodiments, the collection chamber and the heating container are part of a fluid network.

[0224] After release from the autoclave, the autoclaved processed sample can undergo further sample processing or analysis. The autoclaved processed sample can be used for nucleic acid detection and / or analysis using PCR analysis or biological (e.g., diagnostic) assays or any nucleic acid analysis method disclosed elsewhere herein.

[0225] Certain embodiments of the present disclosure

[0226] Embodiment 1. A method for analyzing nucleic acids in a biological sample, the method 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.

[0227] Embodiment 2. A method for analyzing nucleic acids in a biological sample, the method comprising: heating the biological sample to a temperature of at least 101 degrees Celsius in a closed container; and analyzing the nucleic acids.

[0228] Embodiment 3. A method for analyzing nucleic acids in a biological sample, the method comprising: heating the biological sample containing the nucleotides in a closed container, thereby generating a pressure between 1 and 200 PSI exceeding atmospheric pressure in the container; and analyzing the nucleic acids.

[0229] Embodiment 4. A method for analyzing nucleic acids in a biological sample, comprising: heating the biological sample under high pressure conditions; and analyzing the nucleic acids.

[0230] Embodiment 5. A method for improving the efficiency of molecular amplification, comprising: heating a biological sample in a closed container to generate a pressure between 10 and 100 PSI exceeding atmospheric pressure in the container, wherein the biological sample contains nucleic acid.

[0231] Embodiment 6. A method of inactivating a molecular amplification inhibitor in a biological sample containing nucleic acids, the method comprising: heating the biological sample to a temperature above the boiling point of the biological sample, wherein the nucleic acids are substantially not degraded.

[0232] Embodiment 7. A method according to embodiment 5 or embodiment 6, wherein the method further comprises analyzing the nucleic acid.

[0233] Embodiment 8. A method according to any one of embodiments 1 to 7, wherein the analysis of the nucleic acid comprises one or more of the following: detecting the nucleic acid; sequencing the nucleic acid; and genotyping the nucleic acid.

[0234] Embodiment 9. A method according to any one of Embodiments 1 to 8, wherein the method further comprises detecting the nucleic acid by fluorescence detection.

[0235] Embodiment 10. A method according to any one of embodiments 1 to 9, wherein the method further comprises amplifying the nucleic acid using molecular amplification.

[0236] Embodiment 11. A method according to embodiment 10, wherein the method further comprises amplifying the nucleic acid using molecular amplification before analyzing the nucleic acid.

[0237] Embodiment 12. A method according to embodiment 11, wherein the method further comprises amplifying the nucleic acid using molecular amplification after heating the biological sample.

[0238] Embodiment 13. A method for analyzing nucleic acids in a biological sample, comprising: heating the biological sample in a closed container; generating a pressure greater than 1 atm in the closed container; amplifying the nucleic acids using molecular amplification; and analyzing the nucleic acids.

[0239] Embodiment 14. A method for analyzing nucleic acids in a biological sample, comprising: heating the biological sample to a temperature higher than the boiling point of the biological sample under high pressure conditions; amplifying the nucleic acids using molecular amplification; and analyzing the nucleic acids.

[0240] Embodiment 15. A method according to any one of Embodiments 1 to 14, wherein the heating of the biological sample occurs within a first time period.

[0241] Embodiment 16. A method according to embodiment 15, wherein the first time period is between 30 seconds and 3 minutes.

[0242] Embodiment 17. The method of any one of Embodiments 10 to 16, wherein the molecular amplification comprises polymerase chain reaction ("PCR").

[0243] Embodiment 18. A method according to embodiment 17, wherein the analysis of the nucleic acid includes detecting PCR products formed during molecular amplification.

[0244] Embodiment 19. A method according to any one of embodiments 1 to 18, wherein the biological sample further comprises an additive.

[0245] Embodiment 20. A method according to embodiment 19, wherein the additive is selected from a chelating agent, a reducing agent or a nuclease inhibitor.

[0246] Embodiment 21. A method according to embodiment 20, wherein the additive comprises a chelating agent and a reducing agent.

[0247] Embodiment 22. The method of Embodiment 19 or Embodiment 20, wherein the additive is a chelating agent.

[0248] Embodiment 23. The method of any one of Embodiments 20 to 22, wherein the chelating agent is an insoluble chelating agent.

[0249] Embodiment 24. The method of embodiment 23, wherein the insoluble chelating agent comprises Chelex.

[0250] Embodiment 25. The method of any one of Embodiments 20 to 22, wherein the chelating agent is a soluble chelating agent.

[0251] Embodiment 26. The method of embodiment 25, wherein the soluble chelating agent comprises EDTA.

[0252] Embodiment 27. A method according to Embodiment 19 or Embodiment 20, wherein the additive is a reducing agent.

[0253] Embodiment 28. A method according to any one of embodiments 19 to 27, wherein the additive is added to the biological sample prior to the heating step.

[0254] Embodiment 29. A method according to any one of embodiments 1 to 28, wherein the biological sample also contains a single-chain binding protein.

[0255] Embodiment 30. A method according to any one of embodiments 1 to 29, wherein the nucleic acid is analyzed after amplifying the nucleic acid using between 10 and 45 cycles of molecular amplification.

[0256] Embodiment 31. A method according to any one of Embodiments 1 to 30, wherein the nucleic acid is detectable after fewer cycles of molecular amplification than would be required in the absence of heating.

[0257] Embodiment 32. A method according to any one of embodiments 1 to 31, wherein the nucleic acid is detectable after fewer molecular amplification cycles than required for heating the sample at a temperature below the boiling point of the sample for the same time.

[0258] Embodiment 33. A method according to any one of Embodiments 1 to 32, wherein the nucleic acid is detectable after between 20 and 45 cycles of molecular amplification.

[0259] Embodiment 34. A method according to embodiment 33, wherein the nucleic acid is detectable after between 28 and 35 cycles of molecular amplification.

[0260] Embodiment 35. A method according to any one of embodiments 1 to 34, wherein the analysis of the nucleic acid occurs simultaneously with the molecular amplification of the nucleic acid.

[0261] Embodiment 36. A 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 occur sequentially.

[0262] Embodiment 37. A method according to any one of Embodiments 1 to 35, wherein the analysis of the nucleic acid occurs after each molecular amplification cycle.

[0263] Embodiment 38. A method according to any one of Embodiments 1 to 37, wherein the biological sample also contains multiple molecular amplification inhibitors, and at least 70% of the multiple molecular amplification inhibitors are inactivated after heating.

[0264] Embodiment 39. A method according to any one of Embodiments 1 to 37, wherein the biological sample also contains multiple molecular amplification inhibitors, and at least 90% of the multiple molecular amplification inhibitors are inactivated after heating.

[0265] Embodiment 40. The method of any one of Embodiments 1 to 39, wherein the nucleic acid is not substantially degraded after heating.

[0266] Embodiment 41. The method of any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is at least 120 degrees Celsius.

[0267] Embodiment 42. The method of 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.

[0268] Embodiment 43. The method of 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.

[0269] Embodiment 44. The method of any one of Embodiments 1 to 2, 6 to 12, and 14 to 40, wherein the temperature is about 130 degrees Celsius.

[0270] Embodiment 45. The method of any one of Embodiments 1 to 44, wherein the biological sample is heated in a hermetically sealed container.

[0271] Embodiment 46. A method according to embodiment 45, wherein heating the biological sample generates a pressure between 10 and 100 PSI within the hermetically sealed container.

[0272] Embodiment 47. The method of any one of Embodiments 1 to 46, wherein the pH of the biological sample is about 8.0 to about 12.0.

[0273] Embodiment 48. The method of any one of Embodiments 1 to 47, wherein the nucleic acid is DNA.

[0274] Embodiment 49. The method of any one of Embodiments 1 to 47, wherein the nucleic acid is RNA.

[0275] Embodiment 50. The method of any one of embodiments 1 to 49, wherein the nucleic acid is selected from the group consisting of viral nucleic acid, bacterial nucleic acid, protozoan nucleic acid, and fungal nucleic acid.

[0276] Embodiment 51. A method according to embodiment 50, wherein the nucleic acid is a viral nucleic acid.

[0277] Embodiment 52. The method according to any one of embodiments 1 to 51, wherein the biological sample contains 50 copies / mL and 10 9 The nucleic acid was present at a concentration of between 100 copies / mL.

[0278] Embodiment 53. A method according to any one of Embodiments 1 to 52, wherein the volume of the biological sample is between 100 μL and 5 mL.

[0279] Embodiment 54. The method of any one of Embodiments 1 to 53, wherein the biological sample comprises a pathogen or a portion of a pathogen.

[0280] Embodiment 55. The method of embodiment 54, wherein the pathogen or a portion of a pathogen is selected from the group consisting of a virus or a portion of a virus, a bacterium or a portion of a bacterium, a protozoa or a portion of a protozoa, a yeast or a portion of a yeast, and a fungus or a portion of a fungus.

[0281] Embodiment 56. The method of embodiment 55, wherein the pathogen is a virus or a portion of a virus.

[0282] Embodiment 57. A method according to Embodiment 56, wherein the virus is SARS-CoV2.

[0283] Embodiment 58. The method of any one of Embodiments 1 to 57, wherein the biological sample comprises a substance selected from the group consisting of blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine.

[0284] Embodiment 59. A method according to embodiment 58, wherein the biological sample comprises mucus.

[0285] Embodiment 60. The method of embodiment 59, wherein the biological sample is collected from the subject via a nasopharyngeal swab, a cervical swab, or a nasal swab.

[0286] Embodiment 61. A method according to any one of Embodiments 1 to 60, wherein the biological sample is heated with a heat source.

[0287] Embodiment 62. A method according to embodiment 61, wherein the heat source comprises an induction heater, a heating element or a microwave.

[0288] Embodiment 63. A method according to any one of embodiments 1 to 2, 6 to 12, or 14 to 62, wherein the biological sample is maintained at the temperature for between 1 second and 300 seconds.

[0289] Embodiment 64. The method of any one of Embodiments 1 to 2, 6 to 12, or 14 to 62, wherein the biological sample is maintained at the temperature for between 30 seconds and 120 seconds.

[0290] Embodiment 65. A method according to any one of Embodiments 1 to 64, wherein the biological sample is collected from a subject.

[0291] Embodiment 66. The method of embodiment 65, wherein the subject is a human.

[0292] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may conceive of many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0293] Example

[0294] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0295] Example 1: High pressure heating method effectively neutralizes RT-PCR inhibitors

[0296] 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 mean cycle threshold) of the two sample preparation methods was compared.

[0297] Materials used

[0298] Cryotube; Metal paramagnetic galvanized 6 mm dish; 1.5 ml collection buffer consisting of 10 mm Tris / EDTA, 1 mM NaCl; 10% W / v Chelex 100 resin; CDC SARS-COV2 RT-PCR primers; Puritan swab.

[0299] Experimental Procedure

[0300] The nasal specimen was collected using a Plex swab and the nasal matrix was resuspended in 1.5 ml of collection buffer. Next, 250 microliters of the nasal specimen resuspended in collection buffer was placed in a cryogenic tube with two metal disks at the bottom and 10% Chelex 100. 32,000 copies of inactivated SARS-COV2 virus per ml were added to the cryogenic tube.

[0301] For the method of the present disclosure, the cryotube was then placed under an oscillating magnetic field generated by an induction coil and then heated at high pressure for 40 seconds, 60 seconds, 80 seconds and 100 seconds, respectively, to 100° C., 103° C., 108° C. and 110° C. For control, identical samples in the same buffer and using the same Chelex percentage were heated in a heat block at 98° C. for 5 minutes.

[0302] result

[0303] Figure 1 Results of RT-PCR experiments following the conditions described in the experimental procedures (autoclaving at or above 100 degrees Celsius for 40 seconds, 60 seconds, 80 seconds, and 100 seconds) and standard conditions of heating at 98 degrees Celsius for 300 seconds are shown. Each sample preparation method was performed on 10 μL and 20 μL samples to evaluate any effects from sample volume. All tested conditions effectively neutralized PCR inhibitors from nasal specimens, which was indicated by an average cycle threshold of 33 or less. In addition, autoclaving the 10 μL sample to above 100 degrees Celsius for 40 seconds, 60 seconds, and 80 seconds resulted in an average cycle threshold of 31 or less, which was comparable to heating for 300 seconds and 98 degrees Celsius. In addition, for the 20 μL sample, all tested conditions (including autoclaving above 100 degrees Celsius) achieved an average cycle threshold of less than 31. Therefore, adding more volume to the preparation improves the sensitivity of PCR, and PCR inhibitors are not reactivated in the sample preparation.

[0304] Figure 1 The results set forth in show that the methods of the present disclosure give superior results to conventional methods of heating at 95-98 degrees C. For example, 60 seconds of high pressure heating to 103 degrees C significantly outperforms the standard condition of 300 seconds at 98 degrees C, providing a lower average cycle threshold in one-fifth the time.

[0305] Example 2: Effect of sample preparation temperature on RT-PCR efficiency

[0306] In this example, the effect of temperature on sample preparation was tested. RT-PCR was run to determine the efficiency of sample preparation at different temperatures (95°C, 105°C, 120°C and 130°C).

[0307] Materials used

[0308] Glass ampoule; 10% Chelex 100; collection buffer: 10 mm Tris / EDTA, 1 mM NaCl, 0.02% azide; 1 micromolar thermostable single-stranded nucleic acid binding protein; heat block with aluminum beads; CDC Sars-CoV2 RT-PCR primers; QiaAmp viral RNA kit.

[0309] Experimental Procedure

[0310] The heat block was set at 95°C, 105°C, 120°C, and 130°C. 250 microliters of nasal matrix was resuspended in collection buffer and 32,000 copies of inactivated SARS-COV2 were added. The 250 microliter sample was then placed in a glass ampoule and then sealed airtight. The tube was placed in a heat block at the appropriate temperature for 2 minutes. The supernatant was collected and RT-PCR was run on a commercial thermal cycler. For control, the same sample in the same buffer was also tested at the same temperature.

[0311] result

[0312] Figure 2 Depicted are the average cycle thresholds for detection of inactivated SARS-CoV-2 in (1) simple buffer solutions (black dots on the graph) and (2) swab samples. Each of these samples was subjected to a different temperature (95°C, 105°C, 120°C, and 130°C) for 2 minutes.

[0313] Figure 2 The results described in show that the methods of the present disclosure provide effective neutralization of PCR inhibitors. Figure 2 As seen in the swab and buffer samples heated at 95°C, the average cycle thresholds were 36 and 30, respectively. The average cycle thresholds of the swab samples (light gray dots, upper left) were higher than those of the buffer samples (black dots, lower left), indicating that PCR inhibitors remained in the nasal samples after heating and were not present in the buffer solution. Therefore, the standard method of heating at 95°C failed to inactivate all PCR inhibitors in the nasal swab samples after 2 minutes. In contrast, after high-pressure heating at 130°C for 2 minutes, both the nasal swab samples (light gray dots, right) and the buffer samples (black dots, right) exhibited essentially the same average cycle threshold (average cycle threshold of approximately 30), indicating that all PCR inhibitors present in the nasal swab samples were almost completely inactivated.

[0314] also, Figure 2The results depicted indicate that the methods of the present disclosure do not cause detectable nucleic acid degradation. The mean cycle threshold values ​​for the buffer samples were consistent at all temperatures examined (mean cycle threshold value of approximately 30), indicating that autoclaving the buffer samples to temperatures up to 130°C did not cause significant degradation of the nucleic acids in the buffer samples. In addition, no significant degradation was identified in the nasal swab sample (light grey dots, right) as it exhibited the same mean cycle threshold value as the buffer sample (black dots, right). Based on Figure 2 It is apparent that the method of the present disclosure achieves effective inactivation of PCR inhibitors without degrading nucleic acids.

[0315] Example 3: High pressure heating method matches industry standard procedures for time required for PCR amplification

[0316] 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 at different concentrations of analyte (SARS-CoV2 copies).

[0317] Materials used

[0318] Glass ampoule; 10% Chelex 100; collection buffer: 10 mm Tris / EDTA, 1 mM NaCl, 0.02% azide; 1 micromolar thermostable single-stranded nucleic acid binding protein; heat block with aluminum beads; CDC Sars-CoV2 RT-PCR primers; QiaAmp viral RNA kit.

[0319] Experimental Procedure

[0320] Several aliquots of 250 microliters of nasal matrix were resuspended in collection buffer, and different concentrations of inactivated SARS-CoV2 (200, 500, 1000, 2000, 4000, 8000, 16000, and 32000 copies of inactivated SARS-CoV2 per milliliter) were added to each aliquot.

[0321] For the disclosed method (FAST PCR preparation), the heat block was set at 130 degrees Celsius. Each 250 microliter sample was then placed in a glass ampoule and then hermetically sealed. The tube was placed at 130 degrees Celsius for 2 minutes. The supernatant was collected and RT-PCR was run on a commercial thermal cycler.

[0322] At the same time, as a control, a sample aliquot of a second copy of SARS-CoV2 at the same copy / ml concentration was purified using the QiaAmp Viral RNA Kit according to the manufacturer's instructions. Each experimental group was performed twice.

[0323] result

[0324] Figure 3 It was shown that the mean cycle threshold from the method of the present disclosure (labeled "FASTPCR Overheating Preparation") was similar to the mean cycle threshold obtained from the sample preparation using the Qiagen Viral RNA kit. Similar mean cycle threshold values ​​between the two methods were observed at various concentrations of analyte in the nasal specimens, with higher analyte concentrations resulting in lower mean cycle threshold values ​​for each method. Thus, Figure 3 The results described in demonstrate that the PCR efficiency of the disclosed method matches the performance of the Qiagen Viral RNA kit for detecting SARS-COV2 in a significantly shorter time.

[0325] Example 4: High pressure heating method without chelating agent

[0326] In this example, different reagents, such as RNase inhibitors and / or reducing agents, were tested in sample preparation for RT-PCR using the methods of the present disclosure.

[0327] Used Material

[0328] Stainless steel cup; collection buffer including 10 mm Tris / EDTA, 1 mM NaCl; induction heating system; and respiratory syncytial virus (RSV) RT-PCR primers.

[0329] Experimental Procedure

[0330] 250 μL of nasal swab matrix samples were collected and resuspended in collection buffer. 32,000 copies of inactivated respiratory syncytial virus (RSV) were added to the mixture of the collection and nasal swab matrix. Before heating, some aliquots of the sample were: (a) not added to anything, (b) 1 mM DTT was added, (c) only 200 U of RNase (Ambion) was added, or (d) a combination of 1 mM DT and 200 U of RNase was added. The 250 μL sample was then placed in a stainless steel container and then hermetically sealed. Next, the container was placed in an induction heating system set to 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 run on a commercial thermal cycler.

[0331] For control purposes, identical samples were tested in clean buffer using the same conditions.

[0332] result

[0333] Figure 4 Table showing the average cycle thresholds for detection of inactivated RSV RNA in clean buffer and mixed swabs under the following four conditions: (1) no additive (first row); (2) 200U of RNase inhibitor only (second row); (3) 1mM DTT only (third row); and (4) both 1mM DTT and 200U RNase inhibitor (fourth row). In any of these test groups, no chelating agent was added. UD means not detected, and N / A means not applicable.

[0334] The results show that in the absence of Chelex, sample preparation using autoclaved mixed swab matrix was effective at least in the presence of RNase inhibitors or reducing agents such as 1 mM DDT (mean cycle thresholds of 34.9 and 33.1, respectively). The combination of reducing agent and RNase inhibitor in the collection buffer provided even better results for sample preparation of mixed swab matrix using the autoclaved method. The mean cycles of RT-PCR from sample preparations using both reducing agent and RNase inhibitor were similar to control samples using clean buffer (mean cycle thresholds of 30.8 and 28.9, respectively), indicating that the PCR inhibitors in the mixed swab matrix samples were almost completely inactivated.

[0335] according to Figure 4 The results in the literature show that the high pressure heating method of sample preparation provides effective neutralization of PCR inhibitors even in the absence of Chelex. The method is particularly effective in the presence of reducing agents such as DDT, RNase inhibitors, and / or a combination of reducing agents and RNase inhibitors. Additionally, Figure 4 The results presented in show that PCR inhibitors are almost completely neutralized when RNase is added together with a reducing agent.

[0336] Therefore, the high pressure heating method of the present disclosure is effective for sample preparation (e.g., inactivation of PCR inhibitors) even in the absence of a chelating agent, and is particularly effective for sample preparation (e.g., inactivation of PCR inhibitors) in the presence of additives (e.g., reducing agents, RNase inhibitors and / or combinations thereof).

[0337] Example 5: High Pressure Heating Across Multiple Sample Sources

[0338] In this example, the method of the present disclosure was shown to provide similar PCR detection and sensitivity for a variety of nucleic acid-containing samples compared to standard sample preparation methods using the Qiagen Viral RNA kit or the Qiagen DNeasy UltraCleanMicrobe DNA kit.

[0339] Materials used

[0340] Metal heating container; induction coil; collection buffer (1x TE buffer and 2mg / mL BSA); Chelex-100 chelating resin (final concentration in treated sample before autoclaving: 12% weight / volume of treated sample); DTT (final concentration in treated sample before autoclaving: 1mM); thermostable Thermococcus kodakarensis (KOD) (final concentration in treated sample before autoclaving: 0.5μM); nucleic acid-containing sample; centrifuge; PCR primers targeting nucleic acid in nucleic acid-containing sample; Qiagen Viral RNA kit; Qiagen DNeasy UltraClean Microbe DNA kit. The treated sample is a mixture of nucleic acid-containing sample, collection buffer, Chelex-100 chelating resin, Thermococcus kodakarensis (KOD) and DTT.

[0341] Samples containing nucleic acid

[0342] The following nucleic acid-containing samples were tested in this example: SARS-CoV-2, HeLa mammalian cells, Saccharomyces cerevisiae yeast, Bacillus cereus bacterial spores, Bacillus subtilis bacteria, and Candida albicans fungi.

[0343] Experimental Procedure

[0344] First, the nucleic acid-containing sample was prepared by PCR using a high pressure heating method. First, the nucleic acid-containing sample was diluted individually using collection buffer (1x TE buffer and 2 mg / mL BSA). The concentration of the diluted nucleic acid sample in the collection buffer is shown in Figure 5For each sample, a metal heating container with the following reagent ingredients was prepared: Chelex-100 chelating resin (48 mg), DTT (3.2 μL) and Thermococcus kodakarensis (KOD) (8 μL), BSA (2 mg / mL). For each run, a diluted nucleic acid-containing sample (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 lid, placed in an induction coil and heated for 15 seconds. The metal heating container was cooled at ambient temperature until it could be safely manipulated. The metal heating container was placed in a centrifuge and rotated to collect material at the bottom of the container. The metal heating container was then opened and the sample was sucked into a microcentrifuge tube. The microcentrifuge tube was rotated to precipitate the Chelex-100 chelating resin, and the supernatant was collected for PCR analysis.

[0345] At the same time, control samples were also prepared from aliquots of the original diluted nucleic acid samples. These samples were not subjected to autoclaving and were purified using commercial nucleic acid purification kits. 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, Saccharomyces cerevisiae yeast, Bacillus cereus bacterial spores, Bacillus subtilis bacteria, and Candida albicans fungi, nucleic acids were purified using the Qiagen DNeasy UltraClean Microbe DNA Kit according to the manufacturer's instructions. The microbial DNA extraction protocol requires 1.8 mL of sample and is performed for 70 minutes.

[0346] PCR analysis was then performed using samples prepared by autoclaving and control samples prepared by commercial purification kits. PCR amplification was performed on Applied Biosystems Quantstudio 3 and Quantstudio 5 PCR instruments. Each experimental group was performed twice.

[0347] result

[0348] The results show that the average cycle threshold values ​​from the method of the present disclosure (labeled "Autoclave") are similar to the average cycle threshold values ​​obtained from sample preparations using either the Qiagen Viral RNA kit preparation or the Qiagen DNeasy UltraClean Microbe DNA kit across different nucleic acid-containing samples. Figure 5A table showing the average cycle thresholds for detecting inactivated nucleic acid samples from each sample source is shown. 15 seconds of high pressure heating produced equivalent (within the error range) or faster Ct values ​​for each sample. Thus, Figure 5 The results presented in demonstrate that the PCR efficiency of the disclosed method matches the performance of commercially available methods for detecting nucleic acids from multiple sample sources, including SARS-CoV-2, mammalian cells, yeast, bacteria, and fungi, in significantly shorter times.

[0349] Example 6: Modification of temperature ramp rates for nucleic acid sample preparation by high pressure heating

[0350] In this embodiment, different high pressure heating containers are created for high pressure heating with different heating temperature ramp rates. The temperature is estimated using an internal temperature sensor inside the closed container and is correlated by an external temperature infrared sensor that evaluates the external temperature of the closed container. Table 1 shows the different heating containers created and the heating method (high pressure heating time) and heating temperature ramp rate for each container.

[0351] Table 1. High Pressure Heating Temperature Ramp Rates

[0352]

[0353]

[0354] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in the practice of the present invention. It is understood that various alternatives to the embodiments of the present invention described herein may be employed. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for analyzing nucleic acid, the method comprising: (a) heating a treated sample containing nucleic acid from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat treated sample; and (b) analyzing the nucleic acid from the heat-treated sample. 2 . The method according to claim 1 , wherein the treated 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.

3. The method according to claim 1 or 2, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein and a reducing agent.

4. The method according to any one of claims 1 to 3, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent and a stabilizer.

5. The method according to any one of claims 1-4, wherein the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 101 degrees Celsius to 160 degrees Celsius.

6. The method according to any one of claims 1-5, wherein the ramp time is 3 to 50 seconds.

7. The method of any one of claims 1-6, wherein heating the process sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

8. The method according to any one of claims 1 to 7, further comprising after step (a) and before step (b): The treated sample is held at the second temperature for a holding time of 0 seconds to 120 seconds prior to a cool down time.

9. The method of any one of claims 1 to 8, wherein the processed sample comprises a body sample containing the nucleic acid, wherein: 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 stool sample, a cerebrospinal fluid sample, and a urine sample; and ii) not extracting, isolating or otherwise purifying said nucleic acid from said body sample.

10. A method for analyzing nucleic acid, the method 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 stool sample, a cerebrospinal fluid sample, and a urine sample; in The body sample comprises nucleic acid; (b) heating a treated sample comprising the body sample from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (c) analyzing the nucleic acid from the heat-treated sample; wherein the nucleic acid is not extracted, isolated or otherwise purified from the body sample.

11. The method of claim 9 or 10, wherein the body sample is a saliva sample or a mucus sample.

12. The method according to any one of claims 9-11, wherein the body sample is collected from a human subject by nasopharyngeal swab, cervical swab or nasal swab.

13. The method of any one of claims 9-12, wherein the body sample comprises a pathogen or a portion of a pathogen.

14. The method according to claim 13, wherein the pathogen or a part of a pathogen is selected from the group consisting of a virus or a part of a virus, a bacterium or a part of a bacterium, a protozoa or a part of a protozoa, a yeast or a part of a yeast, and a fungus or a part of a fungus.

15. The method according to claim 13 or 14, wherein the pathogen is a virus or a part of a virus.

16. The method of claim 14 or 15, wherein the virus is SARS-CoV2.

17. The method of any one of claims 1-16, wherein the treated sample comprises unlysed cells containing the nucleic acid, and wherein the nucleic acid is not extracted, isolated or otherwise purified from the heat-treated sample prior to analyzing the nucleic acid from the heat-treated sample.

18. A method for analyzing nucleic acid, the method comprising: (a) heating a treated sample comprising unlysed cells containing nucleic acid from a first temperature to a second temperature above 100 degrees Celsius in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; as well as (b) analyzing the nucleic acid from the heat-treated sample; Wherein prior to step (b), the nucleic acid is not extracted, separated or otherwise purified from the heat-treated sample.

19. The method of claim 17 or 18, wherein the unlysed cells are selected from the group consisting of bacterial cells, fungal cells and mammalian cells.

20. The method according to any one of claims 1 to 19, wherein the treated 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.

21. A method for analyzing nucleic acid, the method comprising: (a) heating a treated sample containing nucleic acid from a first temperature to a second temperature above 100 degrees Celsius in a closed heating chamber under high pressure conditions, thereby producing a heat-treated sample; and (b) analyzing the nucleic acid from the heat-treated sample; The treated 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.

22. The method according to claim 20 or 21, wherein the treated sample comprises 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.

23. The method according to any one of claims 20 to 22, wherein the treated sample comprises 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.

24. The method according to any one of claims 20-23, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein and a reducing agent.

25. The method according to any one of claims 20 to 24, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent and a stabilizer.

26. The method according to any one of claims 20-25, wherein the concentration of the chelating agent is 5% to 25% by weight to the volume of the treated sample; the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM; the concentration of the reducing agent in the treated sample is 0.1 mM to 2 mM; or the concentration of the stabilizer in the treated sample is 500 ng / mL to 10 mg / mL.

27. The method of any one of claims 20-26, wherein the chelating agent is an insoluble chelating agent.

28. The method of claim 27, wherein the insoluble chelating agent comprises styrene divinyl benzene copolymer.

29. The method of any one of claims 20-26, wherein the chelating agent is a soluble chelating agent.

30. The method of claim 29, wherein the soluble chelating agent comprises EDTA.

31. The method of any one of claims 20-30, wherein the stabilizer is bovine serum albumin or gelatin.

32. The method of any one of claims 1-31, wherein the nucleic acid is DNA.

33. The method of any one of claims 1-32, wherein the nucleic acid is RNA.

34. The method of any one of claims 1-33, wherein the nucleic acid is selected from the group consisting of viral nucleic acids, bacterial nucleic acids, protozoan nucleic acids, eukaryotic nucleic acids, and fungal nucleic acids.

35. The method of any one of claims 1-34, wherein the nucleic acid is a viral nucleic acid.

36. The method according to any one of claims 1-35, wherein the treated sample contains 50 copies / mL to 10 9 Copies / mL of the nucleic acid.

37. The method of any one of claims 1-36, wherein the volume of the processed sample is 100 μL to 5 mL.

38. The method of any one of claims 1-37, wherein the pH of the treated sample is about 8.0 to about 12.

0.

39. The method of any one of claims 1-38, wherein the treated sample further comprises a protease.

40. The method of any one of claims 1-39, wherein the treated sample further comprises a nuclease inhibitor.

41. The method of any one of claims 1-40, wherein the treated sample is heated with a heat source.

42. The method of claim 41, wherein the heat source comprises an induction heater, a heating element, or microwaves.

43. A method according to claim 41 or 42, wherein the heat source comprises an induction heater.

44. The method of any one of claims 1-43, wherein the enclosed heating chamber comprises a thermally conductive material.

45. The method of any one of claims 1-44, wherein the enclosed heating chamber comprises a ferromagnetic material.

46. ​​The method of any one of claims 1-45, wherein the nucleic acid is not substantially degraded after the heating.

47. The method of any one of claims 1-46, further comprising amplifying the nucleic acid by polymerase chain reaction (PCR) after the heating, thereby producing a PCR product.

48. The method of claim 47, further comprising detecting the PCR product.

49. The method of claim 47 or 48, wherein the PCR product is detectable after fewer cycles of molecular amplification than would be required in the absence of said heating of the treated sample.

50. The method of any one of claims 47-49, wherein the PCR product is detectable after fewer cycles of molecular amplification than required by heating the processed sample at a temperature below 100 degrees Celsius.

51. The method of any one of claims 47-50, wherein the PCR product is detectable after amplifying the nucleic acid using 10 to 55 cycles of molecular amplification.

52. The method of any one of claims 47-51, wherein the PCR product is detectable after 28 to 35 cycles of molecular amplification.

53. The method of any one of claims 1-52, wherein analyzing the nucleic acid comprises detecting the nucleic acid, sequencing the nucleic acid, or genotyping the nucleic acid.

54. The method of any one of claims 1-53, wherein analyzing the nucleic acid comprises detecting the nucleic acid by fluorescence detection.

55. The method of any one of claims 1-54, wherein heating the process sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 20 degrees Celsius per second.

56. A method for inactivating a molecular amplification inhibitor in a processed sample containing nucleic acid, the method comprising: Under high pressure heating conditions, a treated sample containing i) nucleic acid and ii) multiple molecular amplification inhibitors is heated from a first temperature to a second temperature above 100 degrees Celsius within a ramp time in a closed heating chamber, thereby inactivating the molecular amplification inhibitors among the multiple amplification inhibitors and producing a heat-treated sample, wherein the nucleic acid is substantially not degraded.

57. The method of claim 56, wherein the first temperature is 4 degrees Celsius to 40 degrees Celsius, and the second temperature is 101 degrees Celsius to 160 degrees Celsius.

58. The method of claim 56 or 57, further comprising maintaining the treated sample at the second temperature for a holding time prior to a cooling time.

59. The method of claim 58, wherein the holding time is from 0 seconds to 120 seconds.

60. The method of any one of claims 56-59, wherein the method inactivates at least 70% of the plurality of molecular amplification inhibitors.

61. The method of any one of claims 56-60, wherein the method inactivates at least 90% of the plurality of molecular amplification inhibitors.

62. The method of any one of claims 56-61, wherein the processed sample comprises a body sample containing the nucleic acid, wherein: 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 stool sample, a cerebrospinal fluid sample, and a urine sample; and ii) not extracting, isolating or otherwise purifying said nucleic acid from said body sample.

63. The method of claim 62, wherein the body sample is a saliva sample.

64. The method of claim 62, wherein the body sample is a mucus sample.

65. The method of any one of claims 62-64, wherein the body sample is collected from an animal or human subject.

66. The method of any one of claims 62-65, wherein the body sample is collected from the animal or human subject by nasopharyngeal swab, cervical swab, or nasal swab.

67. The method of any one of claims 56-66, wherein the processed sample comprises unlysed cells containing the nucleic acid.

68. The method of any one of claims 56-67, wherein the treated 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.

69. The method according to any one of claims 56-68, wherein the treated sample comprises 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.

70. The method of any one of claims 56-69, wherein the treated sample comprises at least three reagents selected from the group consisting of a chelator, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

71. The method of any one of claims 56-70, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent.

72. The method of any one of claims 56-71, wherein the treated sample comprises a chelating agent, a single-stranded nucleic acid binding protein, a reducing agent, and a stabilizer.

73. The method of any one of claims 56-72, wherein the concentration of the chelating agent is 5% to 25% by weight to the volume of the treated sample; the concentration of the single-stranded nucleic acid binding protein in the treated sample is 0.1 μM to 2 μM; the concentration of the reducing agent in the treated sample is 0.1 mM to 2 mM; or the concentration of the stabilizer in the treated sample is 500 ng / mL to 10 mg / mL.

74. The method of any one of claims 56-73, wherein the pH of the treated sample is about 8.0 to about 12.

0.

75. The method of any one of claims 56-74, wherein the treated sample further comprises a protease.

76. The method of any one of claims 56-75, wherein the treated sample further comprises a nuclease inhibitor.

77. The method of any one of claims 56-76, wherein heating the process sample occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

78. A method for analyzing nucleic acid, the method comprising: (a) heating a treated sample containing nucleic acid from a first temperature to a second temperature greater than 100 degrees Celsius within a ramp time in a closed heating chamber under high pressure conditions, thereby producing a heat treated sample; and (b) analyzing the nucleic acid from the heat-treated sample; Wherein the heating occurs at a temperature ramp rate of 5 degrees Celsius per second to 50 degrees Celsius per second.

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