WO2020143135A1 - 核酸释放剂、核酸pcr扩增方法和pcr扩增试剂盒 - Google Patents

核酸释放剂、核酸pcr扩增方法和pcr扩增试剂盒 Download PDF

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WO2020143135A1
WO2020143135A1 PCT/CN2019/085066 CN2019085066W WO2020143135A1 WO 2020143135 A1 WO2020143135 A1 WO 2020143135A1 CN 2019085066 W CN2019085066 W CN 2019085066W WO 2020143135 A1 WO2020143135 A1 WO 2020143135A1
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nucleic acid
sample
pcr amplification
reaction
releasing agent
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PCT/CN2019/085066
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English (en)
French (fr)
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戴立忠
纪博知
张文曲
邓中平
范文洲
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圣湘生物科技股份有限公司
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Priority to MX2021008299A priority Critical patent/MX2021008299A/es
Priority to EP19908402.1A priority patent/EP3910067B1/en
Priority to ES19908402T priority patent/ES2965345T3/es
Priority to BR112021013524-3A priority patent/BR112021013524A2/pt
Priority to US17/421,390 priority patent/US20220064706A1/en
Publication of WO2020143135A1 publication Critical patent/WO2020143135A1/zh

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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    • C12Y304/21014—Microbial serine proteases (3.4.21.14)

Definitions

  • the invention relates to the field of molecular biology, in particular to a nucleic acid releasing agent, a nucleic acid PCR amplification method and a PCR amplification kit.
  • PCR Polymerase Chain Reaction
  • qPCR real-time fluorescent quantitative PCR
  • RNA sample Because the structure of RNA is single-stranded, unstable and easily degraded, the requirements in the sample processing process are very high, and more complicated methods are required for pre-treatment and nucleic acid extraction and purification of the RNA sample to be purified to obtain pure After the nucleic acid, it can be tested to obtain stable results.
  • a nucleic acid releasing agent including Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol and strong base; wherein, the molar concentration of Tris-HCl is 0.5mM ⁇ 500mM, the molar concentration of sodium chloride is 20mM ⁇ 500mM, the mass concentration of potassium chloride is 5mg/mL ⁇ 8mg/mL, the volume percentage of Tween 20 is 0.1% ⁇ 2%, Triton X-100 The volume percentage is 0.1% ⁇ 3%, the volume percentage of ethylphenyl polyethylene glycol is 0.1% ⁇ 3%, and the mass concentration of strong base is 2mg/mL ⁇ 50mg/mL.
  • the nucleic acid releasing agent of the present invention cleaves cells to release nucleic acids through a certain proportion of strong base, while sodium chloride and potassium chloride protect the nucleic acids by coordinating the ion balance inside and outside the cell membrane.
  • Tris-HCl is used to ensure the pH of cell lysis The value is stable and can be well compatible with the PCR reaction solution during subsequent amplification; Triton X-100 on the one hand can well protect nucleic acids, especially single-stranded RNA, so that the RNA can be alkaline It can be preserved in the environment.
  • Triradon X-100 and potassium chloride can reduce the inhibitory effect of strong alkali environment on the enzyme in the PCR reaction under a certain ratio, thereby ensuring the efficiency of RNA amplification; Tween 20 and B Phenylphenyl polyethylene glycol can protect the reverse transcriptase and make the reverse transcriptase work normally in alkaline environment.
  • the nucleic acid releasing agent of the present invention can make RNA containing samples directly release RNA at room temperature, avoid the problem of sample contamination caused by aerosol caused by heating, and effectively prevent RNA in alkaline environment More importantly, it can be directly mixed with the PCR reaction solution for PCR amplification, and the amplification can be completed without complicated nucleic acid extraction and purification processes. It truly realizes one-room, pollution-free, simple and rapid RNA amplification detection And, the detection sensitivity is high and the repeatability is good.
  • it further includes betaine and bovine serum albumin, and in the nucleic acid releasing agent, the mass concentration of the betaine is 0.1 mg/mL to 20 mg/mL, and the mass of the bovine serum albumin The concentration is 5mg/mL ⁇ 100mg/mL.
  • it further includes proteinase K and lithium dodecyl sulfate, and in the nucleic acid releasing agent, the mass concentration of proteinase K is 0.02 mg/mL to 1.5 mg/mL, and the twelve The mass concentration of lithium alkyl sulfate is 0.4 mg/mL to 30 mg/mL.
  • the invention also provides a nucleic acid PCR amplification method, comprising the steps of: mixing the above nucleic acid releasing agent with a sample, placing it at 25°C to 60°C for 2min to 10min, and then adding a PCR reaction solution to perform PCR amplification.
  • a pretreatment step of the sample is further included: mixing the sample with polyethylene glycol, and then centrifuging to take a precipitate.
  • the conditions for PCR amplification of the enterovirus in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 53°C ⁇ 57°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of the hepatitis C virus in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 58°C ⁇ 62°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of respiratory viruses in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 58°C ⁇ 62°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of respiratory bacteria in the sample are:
  • Amplification reaction 92°C ⁇ 96°C for 8s ⁇ 12s, 58°C ⁇ 62°C for 18s ⁇ 22s, several cycles;
  • the invention also provides a PCR amplification kit, including the nucleic acid releasing agent and the PCR reaction solution.
  • Figure 1 is the real-time fluorescence quantitative PCR amplification curve of Examples 1-25;
  • a nucleic acid releasing agent includes Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, and strong base; wherein, Tris-HCl The molar concentration is 0.5 mM to 500 mM, the molar concentration of sodium chloride is 20 mM to 500 mM, the mass concentration of potassium chloride is 5 mg/mL to 8 mg/mL, the volume percentage of Tween 20 is 0.1% to 2%, Trila The volume percentage of Tong X-100 is 0.1%-3%, the volume percentage of ethylphenyl polyethylene glycol is 0.1%-3%, and the mass concentration of strong base is 2mg/mL-50mg/mL.
  • RNA in the lysate cannot generally be directly detected by PCR amplification.
  • pretreatment methods for PCR amplification detection of RNA samples mainly include boiling cracking method and magnetic bead method.
  • the boiling lysis method is to release the nucleic acid in the sample under the action of the lysis buffer by boiling, dissolve in the lysis buffer, and lyse the cells' proteins and chromosomes while lysing the cells in a boiling water bath. Then, the denatured protein and other impurities are removed by centrifugal precipitation, and the nucleic acid in the supernatant is recovered for PCR amplification.
  • protein solidification causes part of the nucleic acid to be encapsulated and lost along with the centrifugal precipitation, which directly reduces the amount of template nucleic acid in the supernatant and reduces the sensitivity of subsequent amplification detection.
  • the magnetic bead method cells are lysed by the lysis solution, and the free nucleic acid molecules are specifically adsorbed to the surface of the magnetic particles, while impurities such as proteins are not adsorbed but remain in the solution. After a certain time of reaction, the magnetic particles are separated from the liquid under the action of a magnetic field, and then eluted with an eluent to obtain pure nucleic acid.
  • the operation of the magnetic bead method is complicated, and the requirements for the sample size are generally 200 to 600 microliters, and the requirements for instruments and equipment are also very high, which limits its promotion and use.
  • the nucleic acid releasing agent of this embodiment cleaves the cells to release nucleic acids through a certain proportion of strong base, while sodium chloride and potassium chloride protect the nucleic acids by coordinating the ion balance inside and outside the cell membrane.
  • Tris-HCl is used to Ensure that the pH value is stable during cell lysis and is compatible with the PCR reaction solution during subsequent amplification;
  • Triton X-100 can protect nucleic acids, especially single-stranded RNA, on the one hand. RNA can be preserved in alkaline environment.
  • Trilaton X-100 and potassium chloride can reduce the inhibitory effect of strong alkaline environment on enzymes in PCR reaction under a certain ratio, thus ensuring the efficiency of RNA amplification; Tween 20 and ethyl phenyl polyethylene glycol can protect reverse transcriptase and make reverse transcriptase work normally in alkaline environment.
  • the nucleic acid releasing agent can make RNA containing samples directly release RNA at room temperature, avoid the problem of sample contamination caused by heating to generate aerosols, and effectively prevent the degradation of RNA in alkaline environments , And more importantly, it can be directly mixed with the PCR reaction solution for PCR amplification, and the amplification can be completed without complicated nucleic acid extraction and purification processes. It truly realizes one-room, pollution-free, simple and rapid RNA amplification detection, and High detection sensitivity and good repeatability. It can be understood that the nucleic acid releasing agent can be used not only for PCR amplification detection of RNA samples, but also for multiple combined amplification detection of DNA samples or mixed samples of RNA and DNA.
  • the strong base is sodium hydroxide, potassium hydroxide or the like, which can be selected according to needs.
  • the nucleic acid releasing agent further includes betaine and bovine serum albumin, and in the nucleic acid releasing agent, the mass concentration of betaine is 0.1 mg/mL to 20 mg/mL, and the mass concentration of bovine serum albumin is 5 mg/ mL ⁇ 100mg/mL.
  • betaine and bovine serum albumin By adding a certain proportion of betaine and bovine serum albumin, it can cooperate with Triton X-100 to better protect RNA under alkaline conditions, and prevent the denaturation of polymerase and reverse transcriptase, thereby ensuring the rapid release of RNA and Amplify to achieve rapid detection of RNA samples.
  • the nucleic acid releasing agent further includes proteinase K and lithium dodecyl sulfate, and in the nucleic acid releasing agent, the mass concentration of proteinase K is 0.02 mg/mL to 1.5 mg/mL, lithium dodecyl sulfate The mass concentration is 0.4mg/mL ⁇ 30mg/mL.
  • a nucleic acid PCR amplification method includes the following steps: mixing the above nucleic acid releasing agent with a sample, placing it at 25°C to 60°C for 2 min to 10 min, and then adding a PCR reaction solution to perform PCR amplification.
  • the nucleic acid PCR amplification method of this embodiment implements the extraction-free purification and direct amplification operation for nucleic acid samples such as RNA, that is, the above nucleic acid releasing agent is added to the sample to release the nucleic acid from the cells, and then directly added to the PCR reaction solution
  • PCR amplification such as real-time fluorescence quantitative PCR, without the need for boiling heating or extraction and purification, can complete the amplification detection of nucleic acid samples, truly realize one-room, pollution-free, simple and rapid RNA amplification detection, and detection High sensitivity and good repeatability.
  • the volume ratio of the nucleic acid releasing agent to the sample is 1:1 to 5.
  • the sample types include serum, plasma, oropharyngeal swabs, nasopharyngeal swabs, alveolar lavage fluid, feces and other types, which can be directly docked with downstream PCR amplification or gene chips after being mixed with nucleic acid releasing agents Detection method.
  • the PCR reaction solution includes deoxyribonucleoside triphosphate, upstream primers, downstream primers, DNA polymerase, reverse transcriptase, and amplification buffer. It can be understood that the composition of the PCR reaction solution can be selected according to different needs according to the type and purpose of the PCR reaction, but it is not limited thereto. For example, when performing real-time fluorescence quantitative PCR, the PCR reaction solution further includes fluorescent probes or fluorescent dyes.
  • a pretreatment step of the sample is also included: the sample is mixed with a polyethylene glycol (PEG) solution, and then centrifuged to take a precipitate.
  • PEG polyethylene glycol
  • the type of polyethylene glycol is PEG-6000, and the concentration is 0.5% to 5% by volume.
  • virus preservation solution or hemolyzed blood sample containing high salt solution 50-5000 microliters of sample can be taken, PEG solution is added in equal volume, centrifuged at 3000-13000rpm/min for 1-10 minutes, discarded Remove the supernatant and take the precipitate, and then add 50-100 microliters of nucleic acid releasing agent.
  • the conditions for PCR amplification of the enterovirus in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 53°C ⁇ 57°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of hepatitis C virus (HCV) in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 58°C ⁇ 62°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of respiratory viruses in the sample are:
  • Amplification reaction 93°C ⁇ 97°C for 13s ⁇ 17s, 58°C ⁇ 62°C for 28s ⁇ 32s, several cycles;
  • Cooling 23°C ⁇ 27°C for 8s ⁇ 12s.
  • the conditions for PCR amplification of respiratory bacteria in the sample are:
  • Amplification reaction 92°C ⁇ 96°C for 8s ⁇ 12s, 58°C ⁇ 62°C for 18s ⁇ 22s, several cycles;
  • a PCR amplification kit includes the above nucleic acid releasing agent and PCR reaction solution.
  • the PCR amplification kit of this embodiment implements the extraction-free purification of RNA and other nucleic acid samples to directly perform the amplification operation, that is, the above nucleic acid releasing agent is added to the sample to release the nucleic acid from the cells, and then directly added to the PCR reaction solution
  • Perform PCR amplification such as real-time fluorescence quantitative PCR, without the need for boiling heating or extraction and purification, can complete the amplification detection of nucleic acid samples, truly realize one-room, pollution-free, simple and rapid RNA amplification detection, and detection High sensitivity and good repeatability.
  • Examples 1-25 Prepare 25 enterovirus pharyngeal swab samples (viral preservation fluid matrix), take 100 ⁇ L samples at 12000 rpm/min and centrifuge for 10 min, discard the supernatant and add 50 ⁇ L nucleic acid release agent, let stand for 10 min, then Mix 10 ⁇ L and 40 ⁇ L PCR reaction solution for real-time fluorescence quantitative PCR amplification, the amplification curve is shown in Figure 1.
  • the nucleic acid releasing agents used in Examples 1-25 include Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin, Proteinase K, lithium dodecyl sulfate and sodium hydroxide.
  • the molar concentration of Tris-HCl is 0.5 mM
  • the molar concentration of sodium chloride is 500 mM
  • the volume percentage of Tween 20 is 0.1%
  • the volume percentage of Triton X-100 is 3%
  • ethyl phenyl polyethylene The volume percentage of diol is 0.1%
  • the mass concentration of potassium chloride is 8 mg/mL
  • the mass concentration of sodium hydroxide is 2 mg/mL
  • the mass concentration of betaine is 20 mg/mL
  • the mass concentration of bovine serum albumin is 5 mg. /mL
  • the mass concentration of proteinase K is 1.5 mg/mL
  • the mass concentration of lithium dodecyl sulfate is 0.4 mg/mL.
  • Comparative Examples 1-25 The above-mentioned 25 samples were processed by magnetic bead method and subjected to real-time fluorescence quantitative PCR amplification. Comparative Examples 26 to 35: basically the same as Examples 1 to 10, except that the nucleic acid releasing agent does not include Tween 20 and ethylphenyl polyethylene glycol.
  • Comparative Examples 36-45 basically the same as Examples 11-20, except that the nucleic acid releasing agent does not include Triton X-100.
  • Comparative Examples 46 to 50 basically the same as Examples 21 to 25, except that the volume percentage of Triton X-100 in the nucleic acid releasing agent is 8%, and the mass concentration of potassium chloride is 15 mg/mL.
  • Reverse transcription reaction reaction at 50°C for 30min
  • Amplification reaction 95s for 15s, 55s for 30s, 45 cycles;
  • Cooling react at 25°C for 10s.
  • the positive samples of enterovirus can be detected in Comparative Examples 1-25 and Examples 1-25, and the results have a 100% agreement rate and good accuracy, while Comparative Examples 26-50 cannot be stable and successful.
  • a positive sample of enterovirus was detected.
  • the smaller the Ct value the higher the detection sensitivity. Comparing the Ct value, it can be seen that the sensitivity of the embodiment using the nucleic acid releasing agent and nucleic acid PCR amplification method of the present invention in RNA sample amplification detection is comparable to that of the magnetic bead method.
  • Examples 26 to 50 Prepare 25 respiratory sputum samples (saline saline matrix), take 5 ⁇ L of the sample and add 5 ⁇ L of nucleic acid release agent, let stand for 10 min, then add 40 ⁇ L of PCR reaction mixture to perform real-time fluorescence quantitative PCR amplification, amplification The curve is shown in Figure 2.
  • the nucleic acid releasing agents used include Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin, proteinase K, twelve Lithium alkyl sulfate and sodium hydroxide.
  • the molar concentration of Tris-HCl is 500mM
  • the molar concentration of sodium chloride is 20mM
  • the volume percentage of Tween 20 is 2%
  • the volume percentage of Triton X-100 is 0.1%
  • ethylphenyl polyethylene The volume percentage of alcohol is 3%
  • the mass concentration of potassium chloride is 5 mg/mL
  • the mass concentration of sodium hydroxide is 50 mg/mL
  • the mass concentration of betaine is 0.1 mg/mL
  • the mass concentration of bovine serum albumin is 100 mg /mL
  • the mass concentration of proteinase K is 0.02 mg/mL
  • the mass concentration of lithium dodecyl sulfate is 30 mg/mL.
  • Comparative examples 51-75 the magnetic beads method was used to process the above 25 samples and perform real-time fluorescence quantitative PCR amplification.
  • UDG enzyme reaction react at 50°C for 2min
  • Amplification reaction 94s for 10s, 60s for 20s, 45 cycles;
  • the positive samples of the respiratory tract bacteria can be detected in the comparative examples and the examples, and the result has a coincidence rate of 100% and good accuracy.
  • the smaller the Ct value the higher the detection sensitivity. Comparing the Ct value, it can be seen that the sensitivity of the embodiment using the nucleic acid releasing agent and nucleic acid PCR amplification method of the present invention in bacterial multiple amplification detection is comparable to that of the magnetic bead method. It also shows that the nucleic acid releasing agent and nucleic acid PCR amplification method of the present invention are not only applicable to the amplification detection of RNA samples, but also applicable to the amplification detection of DNA samples.
  • Examples 51-70 Prepare 20 HCV serum samples, respectively take 15 ⁇ L samples and add 5 ⁇ L nucleic acid releasing agent, let stand for 10 min, then mix with 30 ⁇ L PCR reaction solution for real-time fluorescence quantitative PCR amplification, the amplification curve is shown in Figure 3 .
  • the nucleic acid releasing agent used includes Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin, and sodium hydroxide.
  • the molar concentration of Tris-HCl is 200mM
  • the molar concentration of sodium chloride is 250mM
  • the volume percentage of Tween 20 is 1%
  • the volume percentage of Triton X-100 is 2%
  • ethylphenyl polyethylene The volume percentage of alcohol is 2%
  • the mass concentration of potassium chloride is 7 mg/mL
  • the mass concentration of sodium hydroxide is 25 mg/mL
  • the mass concentration of betaine is 10 mg/mL
  • the mass concentration of bovine serum albumin is 60 mg/ mL.
  • Examples 91 to 110 using the same HCV serum samples as in Examples 51 to 70, taking 15 ⁇ L of the sample and adding 5 ⁇ L of nucleic acid releasing agent, standing for 10 min, and then mixing with 30 ⁇ L of PCR reaction solution for real-time fluorescence quantitative PCR amplification, using nucleic acids
  • the releasing agent is the same as the nucleic acid releasing agent used in Examples 1-25.
  • the Ct value of each example is shown in Table 3.
  • the conditions for PCR amplification are:
  • Pre-denaturation and enzyme activation react at 95°C for 1min;
  • Amplification reaction 95s for 15s, 60s for 30s, 45 cycles;
  • Cooling react at 25°C for 10s.
  • the embodiments of the nucleic acid releasing agent and nucleic acid PCR amplification method of the present invention can detect positive samples of HCV virus, and have good stability.
  • the detection sensitivity of Examples 51 to 70 is slightly inferior to that of Examples 91 to 100, indicating that the effect of the nucleic acid releasing agents used in Examples 51 to 70 is slightly inferior to that of Examples 91 to 100. Agent.
  • Examples 71-90 Prepare 20 respiratory virus pharyngeal swab samples (saline saline matrix), take 100 ⁇ L samples at 12000 rpm/min and centrifuge for 10 min, discard the supernatant and add 50 ⁇ L nucleic acid release agent, let stand for 10 min, then take 10 ⁇ L Mixed with 40 ⁇ L PCR reaction solution for real-time quantitative PCR amplification, the amplification curve is shown in Figure 4.
  • the nucleic acid releasing agent used includes Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, and sodium hydroxide.
  • the molar concentration of Tris-HCl is 400mM
  • the molar concentration of sodium chloride is 150mM
  • the volume percentage of Tween 20 is 0.8%
  • the volume percentage of Triton X-100 is 1.2%
  • ethylphenyl polyethylene The volume percentage of alcohol is 1.5%
  • the mass concentration of potassium chloride is 6 mg/mL
  • the mass concentration of sodium hydroxide is 15 mg/mL.
  • Examples 111-130 using the same respiratory virus pharyngeal swab samples as in Examples 71-90, taking 100 ⁇ L of the sample at 12000 rpm/min and centrifuging for 10 min, discarding the supernatant and adding 50 ⁇ L of nucleic acid-releasing agent, allowing it to stand for 10 min, then taking 10 ⁇ L and 40 ⁇ L of PCR reaction solution were mixed to perform real-time fluorescence quantitative PCR amplification, and the nucleic acid releasing agent used was the same as that used in Examples 51 to 70.
  • Reverse transcription reaction reaction at 50°C for 30min
  • Amplification reaction 95s for 15s, 60s for 30s, 45 cycles;
  • Cooling react at 25°C for 10s.
  • the embodiments of the nucleic acid releasing agent and nucleic acid PCR amplification method of the present invention can detect positive samples of respiratory viruses and have good stability.
  • the detection sensitivity of Examples 71 to 90 is slightly worse than that of Examples 111 to 130, indicating that the effect of the nucleic acid releasing agent used in Examples 71 to 90 is slightly inferior to that of Examples 111 to 130. Agent.

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Abstract

本发明公开一种核酸释放剂、PCR扩增方法和PCR扩增试剂盒。所述核酸释放剂包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇和强碱；其中，Tris-HCl的摩尔浓度为0.5mM～500mM，氯化钠的摩尔浓度为20mM～500mM，吐温20的体积百分比为0.1％～2％，曲拉通X-100的体积百分比为0.1％～3％，乙基苯基聚乙二醇的体积百分比为0.1％～3％，氯化钾的质量浓度为5mg/mL～8mg/mL，强碱的质量浓度为2mg/mL～50mg/mL。该核酸释放剂可以使含有RNA的样品在室温下直接释放出RNA，且能够与PCR反应液直接混合进行PCR扩增。

Description

核酸释放剂、核酸PCR扩增方法和PCR扩增试剂盒 技术领域
本发明涉及分子生物学领域，特别是涉及一种核酸释放剂、核酸PCR扩增方法和PCR扩增试剂盒。
背景技术
PCR(Polymerase Chain Reaction，聚合酶链式反应)是一种用于扩增特定的核酸片段的分子生物学技术，其最大特点，是能将微量的核酸进行大量的富集和增加，从而达到便于检测微量核酸的目的。其中在医学诊断上常用的PCR方法主要是基于双荧光探针的实时荧光定量PCR(qPCR)方法，利用qPCR方法进行体外诊断的靶标主要包括人基因组DNA、DNA病毒、细菌、真菌和RNA病毒等。而由于RNA的结构为单链结构，不稳定且容易降解，因此在样品的处理过程当中要求非常高，需要较为复杂的方法对待扩增的RNA样品进行前处理和核酸提取并纯化，得到纯净的核酸后，方能进行检测得到稳定的结果。
发明内容
基于此，有必要提供一种可简化RNA样品PCR扩增方法的核酸释放剂。
一种核酸释放剂，包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇和强碱；其中，Tris-HCl的摩尔浓度为0.5mM～500mM，氯化钠的摩尔浓度为20mM～500mM，氯化钾的质量浓度为5mg/mL～8mg/mL，吐温20的体积百分比为0.1％～2％，曲拉通X-100的体积百分比为0.1％～3％，乙基苯基聚乙二醇的体积百分比为0.1％～3％，强碱的质量浓度为2mg/mL～50mg/mL。
本发明的核酸释放剂通过一定比例的强碱使细胞裂解释放核酸，而氯化钠和氯化钾则通过协调细胞膜内外离子平衡来对核酸起保护作用，Tris-HCl用于保证细胞裂解时pH值的稳定且能够较好地与后续扩增时的PCR反应液兼容；曲拉通X-100一方面可以很好地对核酸特别是单链的RNA起到保护作用，使RNA能够在碱性环境中得以保存，另一方面曲拉通X-100和氯化钾在一定配比下可以降低强碱环境对PCR反应中酶的抑制作用，从而保证RNA的扩增效率；吐温20和乙基苯基聚乙二醇可对逆转录酶起到保护作用，使逆转录酶在碱性环境中正常工作。通过上述各组分的协同作用，本发明的核酸释放剂可以使含有RNA的样品在室温下直接释放出RNA，避免因加热产生气溶胶导致的样品污染问题，并有效防止RNA在碱性环境中的降解，更重要的是能够与PCR反应液直接混合进行PCR扩增，无需进行复杂的核 酸提取和纯化过程即可完成扩增，真正实现一室化、无污染、简单快速的RNA扩增检测，而且检测灵敏度高，重复性良好。
在其中一个实施例中，还包括甜菜碱和牛血清白蛋白，且在所述核酸释放剂中，所述甜菜碱的质量浓度为0.1mg/mL～20mg/mL，所述牛血清白蛋白的质量浓度为5mg/mL～100mg/mL。
在其中一个实施例中，还包括蛋白酶K和十二烷基硫酸锂，且在所述核酸释放剂中，所述蛋白酶K的质量浓度为0.02mg/mL～1.5mg/mL，所述十二烷基硫酸锂的质量浓度为0.4mg/mL～30mg/mL。
本发明还提供了一种核酸PCR扩增方法，包括以下步骤：将上述核酸释放剂与样品混合，于25℃～60℃放置2min～10min，然后加入PCR反应液进行PCR扩增。
在其中一个实施例中，在将所述核酸释放剂与样品混合之前，还包括样品的预处理步骤：将样品与聚乙二醇混合，然后离心取沉淀。
在其中一个实施例中，对所述样品中的肠道病毒进行PCR扩增的条件为：
逆转录反应：48℃～52℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，53℃～57℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在其中一个实施例中，对所述样品中的丙型肝炎病毒进行PCR扩增的条件为：
预变性和酶激活：93℃～97℃反应0.9min～1.1min；
逆转录反应：58℃～62℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在其中一个实施例中，对所述样品中的呼吸道病毒进行PCR扩增的条件为：
逆转录反应：48℃～52℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在其中一个实施例中，对所述样品中的呼吸道细菌进行PCR扩增的条件为：
UDG酶反应：48℃～52℃反应1.9min～2.1min；
热变性反应：92℃～96℃反应2.9min～3.1min；
扩增反应：92℃～96℃反应8s～12s，58℃～62℃反应18s～22s，若干个循环；
延伸及荧光采集：73℃～77℃反应18s～22s；
溶解曲线：62℃～75℃。
本发明还提供了一种PCR扩增试剂盒，包括上述核酸释放剂和PCR反应液。
附图说明
图1为实施例1～25的实时荧光定量PCR扩增曲线；
图2为实施例26～50的实时荧光定量PCR扩增曲线；
图3为实施例51～70的实时荧光定量PCR扩增曲线；
图4为实施例71～90的实时荧光定量PCR扩增曲线。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，并给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明一实施例的核酸释放剂，包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇和强碱；其中，Tris-HCl的摩尔浓度为0.5mM～500mM，氯化钠的摩尔浓度为20mM～500mM，氯化钾的质量浓度为5mg/mL～8mg/mL，吐温20的体积百分比为0.1％～2％，曲拉通X-100的体积百分比为0.1％～3％，乙基苯基聚乙二醇的体积百分比为0.1％～3％，强碱的质量浓度为2mg/mL～50mg/mL。
为了达到裂解的效果，裂解液需要呈碱性，而RNA在碱性环境中极易被降解，同时针对RNA的PCR扩增检测需要用到逆转录酶，相对于热稳定性较好的TaqDNA聚合酶，逆转录酶较为脆弱，在碱性环境中容易被抑制，无法达到逆转录的效果。因此，RNA在裂解液中一般无法直接进行PCR扩增检测。目前针对RNA样品的PCR扩增检测的前处理方法主要有煮沸裂解法和磁珠法。煮沸裂解法是通过煮沸使样品中的核酸在裂解缓冲液的作用下释放出来，溶解在裂解缓冲液中，沸水浴裂解细胞的同时，使细胞的蛋白质与染色体变性。然后通过离心沉淀去除变性的蛋白质和其他杂质，回收上清液中的核酸用于PCR扩增。然而，经高温煮 沸，蛋白凝固使部分核酸被包裹并随离心沉淀丢失，直接导致上清液中模板核酸量减少，使后续扩增检测灵敏度下降。同时，煮沸加热极容易产生气溶胶，导致样品污染，从而在后续的检测中产生假阳性的结果。磁珠法是通过裂解液裂解细胞，游离出来的核酸分子被特异的吸附到磁性颗粒表面，而蛋白质等杂质不被吸附而留在溶液中。反应一定时间之后，再在磁场作用下，使磁性颗粒与液体分开，再用洗脱液洗脱，得到纯净的核酸。但磁珠法操作复杂，且对样本量的要求一般在200～600微升，仪器设备的要求也非常高，限制了其推广和使用。
针对上述机理，本实施例的核酸释放剂通过一定比例的强碱使细胞裂解释放核酸，而氯化钠和氯化钾则通过协调细胞膜内外离子平衡来对核酸起保护作用，Tris-HCl用于保证细胞裂解时pH值的稳定且能够较好地与后续扩增时的PCR反应液兼容；曲拉通X-100一方面可以很好地对核酸特别是单链的RNA起到保护作用，使RNA能够在碱性环境中得以保存，另一方面曲拉通X-100和氯化钾在一定配比下可以降低强碱环境对PCR反应中酶的抑制作用，从而保证RNA的扩增效率；吐温20和乙基苯基聚乙二醇可对逆转录酶起到保护作用，使逆转录酶在碱性环境中正常工作。通过上述各组分的协同作用，该核酸释放剂可以使含有RNA的样品在室温下直接释放出RNA，避免因加热产生气溶胶导致的样品污染问题，并有效防止RNA在碱性环境中的降解，更重要的是能够与PCR反应液直接混合进行PCR扩增，无需进行复杂的核酸提取和纯化过程即可完成扩增，真正实现一室化、无污染、简单快速的RNA扩增检测，而且检测灵敏度高，重复性良好。可以理解，该核酸释放剂不仅可用于RNA样品的PCR扩增检测，也适用于DNA样品或RNA、DNA混合样品的多重联合扩增检测。
具体地，强碱为氢氧化钠或氢氧化钾等，可根据需要选择。
在一个具体示例中，核酸释放剂还包括甜菜碱和牛血清白蛋白，且在核酸释放剂中，甜菜碱的质量浓度为0.1mg/mL～20mg/mL，牛血清白蛋白的质量浓度为5mg/mL～100mg/mL。通过添加一定比例的甜菜碱和牛血清白蛋白，可以协同曲拉通X-100更好地在碱性条件下对RNA进行保护，以及防止聚合酶和逆转录酶变性，从而确保RNA的快速释放和扩增，实现RNA样品的快速检测。
在一个具体示例中，核酸释放剂还包括蛋白酶K和十二烷基硫酸锂，且在核酸释放剂中，蛋白酶K的质量浓度为0.02mg/mL～1.5mg/mL，十二烷基硫酸锂的质量浓度为0.4mg/mL～30mg/mL。通过添加一定比例的蛋白酶K和十二烷基硫酸锂，可以变性降解RNase，从而进一步保护RNA防止其降解。
本发明一实施例的核酸PCR扩增方法，包括以下步骤：将上述核酸释放剂与样品混合，于25℃～60℃放置2min～10min，然后加入PCR反应液进行PCR扩增。
本实施例的核酸PCR扩增方法针对RNA等核酸样品实现了免提取纯化直接进行扩增的 操作，即将上述核酸释放剂加入到样品中，使核酸从细胞中释放出来，然后直接加入PCR反应液进行PCR扩增例如实时荧光定量PCR，无需进行煮沸加热或提取纯化等过程，就能够完成对核酸样品的扩增检测，真正实现一室化、无污染、简单快速的RNA扩增检测，而且检测灵敏度高，重复性良好。
在一个具体示例中，核酸释放剂与样品的体积比为1∶1～5。可选地，样品类型包括血清、血浆、口咽拭子、鼻咽拭子、肺泡灌洗液、粪便等多种类型，与核酸释放剂混合后可以直接对接下游的PCR扩增或基因芯片等检测方法。
在一个具体示例中，PCR反应液包括脱氧核糖核苷三磷酸、上游引物、下游引物、DNA聚合酶、逆转录酶和扩增缓冲液等。可以理解，根据PCR反应类型和目的的不同，PCR反应液的组成可以根据需要进行选择，不限于此，例如进行实时荧光定量PCR时，PCR反应液还包括荧光探针或荧光染料等。
在一个具体示例中，在将核酸释放剂与样品混合之前，还包括样品的预处理步骤：将样品与聚乙二醇(PEG)溶液混合，然后离心取沉淀。具体地，聚乙二醇的种类为PEG-6000，浓度为体积百分比0.5％～5％。将聚乙二醇作为核酸沉降剂，可以用于处理复杂样品，对于以游离形式存在的RNA病毒，能够有效捕获，增加后期的检测灵敏度，同时不影响PCR的反应体系，可以显著提高RNA快速PCR检测的性能。例如对于含有高盐溶液的细胞保存液、病毒保存液或已经溶血的血液样品等，可取50～5000微升样品，等体积加入PEG溶液，以3000～13000rpm/min进行离心1～10分钟，弃去上清取沉淀，然后再加入50～100微升的核酸释放剂。
在一个具体示例中，对样品中的肠道病毒进行PCR扩增的条件为：
逆转录反应：48℃～52℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，53℃～57℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在一个具体示例中，对样品中的丙型肝炎病毒(HCV)进行PCR扩增的条件为：
预变性和酶激活：93℃～97℃反应0.9min～1.1min；
逆转录反应：58℃～62℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在一个具体示例中，对样品中的呼吸道病毒进行PCR扩增的条件为：
逆转录反应：48℃～52℃反应28min～32min；
热变性反应：93℃～97℃反应0.9min～1.1min；
扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
冷却：23℃～27℃反应8s～12s。
在一个具体示例中，对样品中的呼吸道细菌进行PCR扩增的条件为：
UDG酶反应：48℃～52℃反应1.9min～2.1min；
热变性反应：92℃～96℃反应2.9min～3.1min；
扩增反应：92℃～96℃反应8s～12s，58℃～62℃反应18s～22s，若干个循环；
延伸及荧光采集：73℃～77℃反应18s～22s；
溶解曲线：62℃～75℃。
可以理解，PCR扩增的条件不限于上述具体示例，根据不同的PCR类型和目的可进行相应调整。
本发明一实施例的PCR扩增试剂盒，包括上述核酸释放剂和PCR反应液。本实施例的PCR扩增试剂盒针对RNA等核酸样品实现了免提取纯化直接进行扩增的操作，即将上述核酸释放剂加入到样品中，使核酸从细胞中释放出来，然后直接加入PCR反应液进行PCR扩增例如实时荧光定量PCR，无需进行煮沸加热或提取纯化等过程，就能够完成对核酸样品的扩增检测，真正实现一室化、无污染、简单快速的RNA扩增检测，而且检测灵敏度高，重复性良好。
以下为具体实施例。
一、肠道病毒检测
实施例1～25：准备25份肠道病毒咽拭子样品(病毒保存液基质)，分别取100μL样品于12000rpm/min离心10min，弃去上清并加入50μL核酸释放剂，静置10min，然后取10μL与40μL PCR反应液混合进行实时荧光定量PCR扩增，扩增曲线如图1所示。实施例1～25所用核酸释放剂包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇、甜菜碱、牛血清白蛋白、蛋白酶K、十二烷基硫酸锂和氢氧化钠。其中，Tris-HCl的摩尔浓度为0.5mM，氯化钠的摩尔浓度为500mM，吐温20的体积百分比为0.1％，曲拉通X-100的体积百分比为3％，乙基苯基聚乙二醇的体积百分比为0.1％，氯化钾的质量浓度为8mg/mL，氢氧化钠的质量浓度为2mg/mL，甜菜碱的质量浓度为20mg/mL，牛血清白蛋白的质量浓度为5mg/mL，蛋白酶K的质量浓度为1.5mg/mL，十二烷基硫酸锂的质量浓度为0.4mg/mL。
对比例1～25：采用磁珠法对上述25份样品进行处理并进行实时荧光定量PCR扩增。对比例26～35：与实施例1～10基本相同，区别在于核酸释放剂中不包括吐温20和乙基苯基聚 乙二醇。
对比例36～45：与实施例11～20基本相同，区别在于核酸释放剂中不包括曲拉通X-100。对比例46～50：与实施例21～25基本相同，区别在于核酸释放剂中曲拉通X-100的体积百分比为8％，氯化钾的质量浓度为15mg/mL。
各实施例和对比例的Ct值如表1所示，PCR扩增的条件为：
逆转录反应：50℃反应30min；
热变性反应：95℃反应1min；
扩增反应：95℃反应15s，55℃反应30s，45个循环；
冷却：25℃反应10s。
表1
  对比例1～13 实施例1～13   对比例14～25 实施例14～25
样本1 24.49 24.07 样本14 20.06 21.68
样本2 25.88 25.32 样本15 24.21 20.64
样本3 25.55 23.61 样本16 23.30 23.63
样本4 23.89 23.55 样本17 26.39 27.63
样本5 27.36 26.06 样本18 29.66 28.03
样本6 26.61 28.12 样本19 24.24 21.44
样本7 28.56 29.06 样本20 34.01 34.23
样本8 44.09 37.96 样本21 28.69 27.57
样本9 32.44 31.82 样本22 27.10 25.31
样本10 30.04 29.27 样本23 29.63 31.11
样本11 22.54 24.24 样本24 31.99 32.50
样本12 24.18 25.83 样本25 27.69 27.60
样本13 31.13 29.93      
根据检测结果可知，对比例1～25和实施例1～25均能检测出肠道病毒的阳性样本，结果一致率达100％，准确性较好，而对比例26～50则无法稳定成功地检测出肠道病毒的阳性样本。另外Ct数值越小说明检测灵敏度越高，对比Ct值可知采用本发明核酸释放剂及核酸PCR扩增方法的实施例在RNA样品扩增检测中的灵敏度与磁珠法的灵敏度相当。
二、呼吸道细菌联检
实施例26～50：准备25份呼吸道痰液样本(生理盐水基质)，分别取5μL样品加入5μL核酸释放剂，静置10min，然后加入40μL PCR反应液混合进行实时荧光定量PCR扩增，扩 增曲线如图2所示。所用核酸释放剂包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇、甜菜碱、牛血清白蛋白、蛋白酶K、十二烷基硫酸锂和氢氧化钠。其中，Tris-HCl的摩尔浓度为500mM，氯化钠的摩尔浓度为20mM，吐温20的体积百分比为2％，曲拉通X-100的体积百分比为0.1％，乙基苯基聚乙二醇的体积百分比为3％，氯化钾的质量浓度为5mg/mL，氢氧化钠的质量浓度为50mg/mL，甜菜碱的质量浓度为0.1mg/mL，牛血清白蛋白的质量浓度为100mg/mL，蛋白酶K的质量浓度为0.02mg/mL，十二烷基硫酸锂的质量浓度为30mg/mL。
对比例51～75：采用磁珠法对上述25份样品进行处理并进行实时荧光定量PCR扩增。
各实施例和对比例的Ct值如表2所示，PCR扩增的条件为：
UDG酶反应：50℃反应2min；
热变性反应：94℃反应3min；
扩增反应：94℃反应10s，60℃反应20s，45个循环；
延伸及荧光采集：75℃反应20s；
溶解曲线：62℃～75℃。
表2
  对比例51～63 实施例26～38   对比例64～75 实施例39～50
样本1 35.36 31.34 样本14 23.35 24.14
样本2 29.47 24.54 样本15 36.03 32.69
样本3 30.18 31.1 样本16 36.2 35.99
样本4 37.23 36.56 样本17 36.02 35.95
样本5 33.21 28.44 样本18 33.44 33.2
样本6 27.94 25.69 样本19 38.34 38.32
样本7 26.58 24.945 样本20 30.76 29.145
样本8 31.19 30.495 样本21 36.49 30.85
样本9 33.64 29.675 样本22 26.22 25.925
样本10 37.95 34.455 样本23 23.01 22.96
样本11 32.68 24.13 样本24 31.39 27.32
样本12 27.58 23.355 样本25 29.26 24.015
样本13 34.14 32.605      
根据检测结果可知，对比例和实施例均能检测出呼吸道细菌的阳性样本，结果一致率达100％，准确性较好。另外Ct数值越小说明检测灵敏度越高，对比Ct值可知采用本发明核酸 释放剂及核酸PCR扩增方法的实施例在细菌多重扩增检测中的灵敏度与磁珠法的灵敏度相当。也说明本发明的核酸释放剂及核酸PCR扩增方法不仅仅适用于RNA样品的扩增检测，同时也可应用于DNA样品的扩增检测。
三、HCV病毒检测
实施例51～70：准备20份HCV血清样品，分别取15μL样品加入5μL核酸释放剂，静置10min，然后与30μL PCR反应液混合进行实时荧光定量PCR扩增，扩增曲线如图3所示。所用核酸释放剂包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇、甜菜碱、牛血清白蛋白和氢氧化钠。其中，Tris-HCl的摩尔浓度为200mM，氯化钠的摩尔浓度为250mM，吐温20的体积百分比为1％，曲拉通X-100的体积百分比为2％，乙基苯基聚乙二醇的体积百分比为2％，氯化钾的质量浓度为7mg/mL，氢氧化钠的质量浓度为25mg/mL，甜菜碱的质量浓度为10mg/mL，牛血清白蛋白的质量浓度为60mg/mL。
实施例91～110：使用与实施例51～70相同的HCV血清样品，分别取15μL样品加入5μL核酸释放剂，静置10min，然后与30μL PCR反应液混合进行实时荧光定量PCR扩增，所用核酸释放剂与实施例1～25所用核酸释放剂相同。
各实施例的Ct值如表3所示，PCR扩增的条件为：
预变性和酶激活：95℃反应1min；
逆转录反应：60℃反应30min；
热变性反应：95℃反应1min；
扩增反应：95℃反应15s，60℃反应30s，45个循环；
冷却：25℃反应10s。
表3
  实施例91～100 实施例51～60   实施例101～110 实施例61～70
样本1 30.25 31.32 样本11 24.56 25.13
样本2 27.66 29.78 样本12 25.60 26.03
样本3 28.31 30.28 样本13 27.06 27.92
样本4 29.00 31.23 样本14 27.95 28.88
样本5 28.63 31.81 样本15 31.05 31.64
样本6 27.25 27.94 样本16 28.60 30.55
样本7 27.21 26.85 样本17 29.67 30.76
样本8 29.29 31.27 样本18 30.44 31.49
样本9 28.63 31.64 样本19 19.78 20.25
样本10 30.55 31.95 样本20 33.20 32.01
根据图3可知，采用本发明核酸释放剂及核酸PCR扩增方法的实施例均能检测出HCV病毒的阳性样本，稳定性较好。另外，从表3可以看到实施例51～70的检测灵敏度略差于实施例91～100，说明实施例51～70采用的核酸释放剂的效果略逊于实施例91～100采用的核酸释放剂。
四、呼吸道病毒检测
实施例71～90：准备20份呼吸道病毒咽拭子样品(生理盐水基质)，分别取100μL样品于12000rpm/min离心10min，弃去上清并加入50μL核酸释放剂，静置10min，然后取10μL与40μL PCR反应液混合进行实时荧光定量PCR扩增，扩增曲线如图4所示。所用核酸释放剂包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇和氢氧化钠。其中，Tris-HCl的摩尔浓度为400mM，氯化钠的摩尔浓度为150mM，吐温20的体积百分比为0.8％，曲拉通X-100的体积百分比为1.2％，乙基苯基聚乙二醇的体积百分比为1.5％，氯化钾的质量浓度为6mg/mL，氢氧化钠的质量浓度为15mg/mL。
实施例111～130：使用与实施例71～90相同的呼吸道病毒咽拭子样品，分别取100μL样品于12000rpm/min离心10min，弃去上清并加入50μL核酸释放剂，静置10min，然后取10μL与40μL PCR反应液混合进行实时荧光定量PCR扩增，所用核酸释放剂与实施例51～70所用核酸释放剂相同。
各实施例的Ct值如表4所示，PCR扩增的条件为：
逆转录反应：50℃反应30min；
热变性反应：95℃反应1min；
扩增反应：95℃反应15s，60℃反应30s，45个循环；
冷却：25℃反应10s。
表4
  实施例111～120 实施例71～80   实施例121～130 实施例81～90
样本1 23.64 26.05 样本11 27.56 28.69
样本2 25.97 28.77 样本12 28.67 29.61
样本3 29.55 31.28 样本13 26.22 28.32
样本4 30.52 32.65 样本14 27.88 29.78
样本5 28.10 28.55 样本15 31.36 31.54
样本6 31.69 34.12 样本16 30.69 33.69
样本7 25.19 26.12 样本17 29.89 29.47
样本8 30.26 32.36 样本18 30.79 32.34
样本9 28.96 31.59 样本19 29.20 32.69
样本10 31.60 31.35 样本20 33.59 33.06
根据图4可知，采用本发明核酸释放剂及核酸PCR扩增方法的实施例均能检测出呼吸道病毒的阳性样本，稳定性较好。另外，从表4可以看到实施例71～90的检测灵敏度略差于实施例111～130，说明实施例71～90采用的核酸释放剂的效果略逊于实施例111～130采用的核酸释放剂。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种核酸释放剂，其特征在于，包括Tris-HCl、氯化钠、氯化钾、吐温20、曲拉通X-100、乙基苯基聚乙二醇和强碱；其中，Tris-HCl的摩尔浓度为0.5mM～500mM，氯化钠的摩尔浓度为20mM～500mM，氯化钾的质量浓度为5mg/mL～8mg/mL，吐温20的体积百分比为0.1％～2％，曲拉通X-100的体积百分比为0.1％～3％，乙基苯基聚乙二醇的体积百分比为0.1％～3％，强碱的质量浓度为2mg/mL～50mg/mL。
  2. 根据权利要求1所述的核酸释放剂，其特征在于，还包括甜菜碱和牛血清白蛋白，且在所述核酸释放剂中，所述甜菜碱的质量浓度为0.1mg/mL～20mg/mL，所述牛血清白蛋白的质量浓度为5mg/mL～100mg/mL。
  3. 根据权利要求1所述的核酸释放剂，其特征在于，还包括蛋白酶K和十二烷基硫酸锂，且在所述核酸释放剂中，所述蛋白酶K的质量浓度为0.02mg/mL～1.5mg/mL，所述十二烷基硫酸锂的质量浓度为0.4mg/mL～30mg/mL。
  4. 一种核酸PCR扩增方法，其特征在于，包括以下步骤：将权利要求1～3任一项所述的核酸释放剂与样品混合，于25℃～60℃放置2min～10min，然后加入PCR反应液进行PCR扩增。
  5. 根据权利要求4所述的核酸PCR扩增方法，其特征在于，在将所述核酸释放剂与样品混合之前，还包括样品的预处理步骤：将样品与聚乙二醇混合，然后离心取沉淀。
  6. 根据权利要求4所述的核酸PCR扩增方法，其特征在于，对所述样品中的肠道病毒进行PCR扩增的条件为：
    逆转录反应：48℃～52℃反应28min～32min；
    热变性反应：93℃～97℃反应0.9min～1.1min；
    扩增反应：93℃～97℃反应13s～17s，53℃～57℃反应28s～32s，若干个循环；
    冷却：23℃～27℃反应8s～12s。
  7. 根据权利要求4所述的核酸PCR扩增方法，其特征在于，对所述样品中的丙型肝炎病毒进行PCR扩增的条件为：
    预变性和酶激活：93℃～97℃反应0.9min～1.1min；
    逆转录反应：58℃～62℃反应28min～32min；
    热变性反应：93℃～97℃反应0.9min～1.1min；
    扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
    冷却：23℃～27℃反应8s～12s。
  8. 根据权利要求4所述的核酸PCR扩增方法，其特征在于，对所述样品中的呼吸道病 毒进行PCR扩增的条件为：
    逆转录反应：48℃～52℃反应28min～32min；
    热变性反应：93℃～97℃反应0.9min～1.1min；
    扩增反应：93℃～97℃反应13s～17s，58℃～62℃反应28s～32s，若干个循环；
    冷却：23℃～27℃反应8s～12s。
  9. 根据权利要求4所述的核酸PCR扩增方法，其特征在于，对所述样品中的呼吸道细菌进行PCR扩增的条件为：
    UDG酶反应：48℃～52℃反应1.9min～2.1min；
    热变性反应：92℃～96℃反应2.9min～3.1min；
    扩增反应：92℃～96℃反应8s～12s，58℃～62℃反应18s～22s，若干个循环；
    延伸及荧光采集：73℃～77℃反应18s～22s；
    溶解曲线：62℃～75℃。
  10. 一种PCR扩增试剂盒，其特征在于，包括权利要求1～3任一项所述的核酸释放剂和PCR反应液。
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