WO2021174673A1 - 病毒核酸检测的预处理方法、预处理液、试剂盒及其用途 - Google Patents

病毒核酸检测的预处理方法、预处理液、试剂盒及其用途 Download PDF

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WO2021174673A1
WO2021174673A1 PCT/CN2020/090052 CN2020090052W WO2021174673A1 WO 2021174673 A1 WO2021174673 A1 WO 2021174673A1 CN 2020090052 W CN2020090052 W CN 2020090052W WO 2021174673 A1 WO2021174673 A1 WO 2021174673A1
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concentration
pretreatment
virus
nucleic acid
detection
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French (fr)
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戴立忠
纪博知
邓中平
刘佳
谭德勇
范旭
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圣湘生物科技股份有限公司
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Priority to EP20923230.5A priority Critical patent/EP4006169A4/en
Priority to BR112022017631A priority patent/BR112022017631A2/pt
Publication of WO2021174673A1 publication Critical patent/WO2021174673A1/zh
Priority to US17/891,404 priority patent/US20220389482A1/en
Priority to ZA2022/10220A priority patent/ZA202210220B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

  • the present invention relates to the field of nucleic acid detection of virus samples; in particular, it relates to a virus sample pretreatment method and a pretreatment solution, which are used for pretreatment of DNA/RNA viruses.
  • the current traditional virus pretreatment solution mainly has two methods: Hank's matrix-based pretreatment solution and guanidine salt-based pretreatment solution.
  • Hank's solution is a common Balanced Salt Solution (BSS) for virus delivery.
  • BSS Balanced Salt Solution
  • Hank's solution can only store respiratory viruses (such as influenza virus, new coronavirus, etc.) for a few hours. After a few hours, the virus shape will be affected, which will affect the efficiency of virus nucleic acid detection. If the time is longer, bacteria and fungi will easily grow, which will cause the pH of Hank's solution to drop and accelerate virus degradation, so it is difficult to use for long-term virus storage and nucleic acid detection.
  • the concentration of guanidine hydrochloride or Rnasin in the guanidine salt-based virus pretreatment solution is relatively high (3-5M), and the high concentration of guanidine salt is suitable for virus inactivation at room temperature.
  • the guanidine salt concentration is too high, it has a great inhibitory effect on nucleic acid extraction or purification such as the magnetic bead method. It requires multiple washings or spin columns to proceed.
  • Extraction Free Nucleic Acid Release and Amplification Technology refers to the direct use of strong bases without the need for nucleic acid extraction or purification of samples Under the nature of the sample nucleic acid release agent and high compatibility amplification system, direct sample nucleic acid amplification detection. Such a “one-step” operation will greatly save the detection time of viral nucleic acid detection, especially RNA virus detection. Compared with the traditional amplification method after nucleic acid extraction, this invention can save more than 60% of the time and increase the detection efficiency by 50%.
  • the present invention provides a pretreatment method for virus nucleic acid detection.
  • the method includes mixing a sample stored in a pretreatment solution with a nucleic acid release agent and a qPCR amplification reagent,
  • the pretreatment liquid includes: Tris-HCl, EDTA-2Na, sodium chloride, ribonuclease inhibitors and antibiotics;
  • the pH of the pretreatment liquid is 6.5-8.0.
  • Tris-HCl may be present at a concentration of about 10 to about 200 mM, preferably at a concentration of about 80 to about 120 mM, and most preferably at a concentration of about 100 mM.
  • EDTA-2Na may be present at a concentration of about 8 to about 50 mM, preferably at a concentration of about 10 to about 15 mM, and most preferably at a concentration of about 10 mM.
  • sodium chloride may be present at a concentration of about 0.5% to about 2% (w/v), preferably at a concentration of about 0.8 to about 1% (w/v), and most preferably at a concentration of about 0.9% (w/v) The concentration of /v) exists.
  • ribonuclease inhibitor refers to a chemical substance capable of inhibiting ribonuclease and inactivating it. Including but not limited to diethyl pyrocarbonate (DEPC), RNase protein inhibitor (RNasin), vanadyl ribonucleoside complex, SDS, etc.
  • DEPC diethyl pyrocarbonate
  • RNase protein inhibitor RNase protein inhibitor
  • SDS vanadyl ribonucleoside complex
  • SDS vanadyl ribonucleoside complex
  • the ribonuclease inhibitor may be present at a concentration of about 2U/mL to about 800U/mL, for example, about 40U/mL, about 50U/mL, about 100U/mL; preferably, It is present at a concentration of about 10 to about 30 U/mL; most preferably, it is present at a concentration of 20 U/mL.
  • the pH value is in the range of 6.5 to 8.0, preferably 7.0 to 8.0, and most preferably 7.5.
  • Antibiotics include but are not limited to Proclin antibiotics (such as Proclin 300 and Proclin 950) and NaN 3 .
  • Proclin 300 when used as an antibiotic, its concentration can be about 0.01% (v/v); for another example, when Proclin 950 is used as an antibiotic, its concentration can be about 0.04% (v/v) ; But the present invention is not limited to this.
  • the pretreatment solution is Tris-HCl with a concentration of 100 mM, EDTA-2Na with a concentration of 10 mM, sodium chloride with a concentration of 0.9% (w/v), and a ribonuclease inhibitor.
  • the concentration is 20U/mL, and the concentration of Proclin 950 is 0.04%(v/v);
  • sample refers to a sample that may contain a virus.
  • the sample may be derived from human or animal blood, feces, urine, oral epithelial cells, exfoliated cells, buccal swabs, throat swabs, and the like.
  • the "pretreatment” mentioned in the present invention refers to the processing before the detection of the sample, especially the processing before the nucleic acid detection (based on the one-step method) is performed on the sample.
  • pretreatment liquid refers to a liquid used for pretreatment of a virus sample.
  • the virus may be a DNA virus or an RNA virus.
  • the virus is an RNA virus.
  • the virus is a coronavirus (such as the 2019 novel coronavirus), respiratory syncytial virus, and enterovirus.
  • the nucleic acid detection reaction solution configured by using the pretreatment method of the present invention can directly perform qPCR without an extraction or purification process, which improves the detection efficiency and shortens the detection time.
  • the antibiotics and ribonuclease inhibitors used in the invention can be used for the pretreatment of DNA viruses and RNA viruses, and can prevent the growth of bacteria and other microorganisms from affecting the virus preservation activity under normal temperature conditions. RNA viruses are more easily degraded due to the ubiquitous RNase.
  • the method of the present invention is used to avoid the medical sampling process (oral epithelial cells, exfoliated cells, throat swabs and other sample types) due to the sample itself or sampling consumables. The damage to RNA caused by itself is very conducive to the long-term preservation and detection of RNA viruses.
  • the composition of the pretreatment liquid of the present invention Points have the effect of enhancing RT-PCT.
  • the present invention provides a method for detecting viral nucleic acid in a sample, which includes directly performing qPCR amplification and detection using the reaction solution configured in the foregoing method.
  • a sample release agent refers to a chemical reagent capable of releasing nucleic acid in a sample.
  • strong acid or strong alkaline chemical reagents may include 0.01-0.5 mmol/L surfactin, 100-200 mmol/L potassium chloride, 50-200 mmol/L lithium chloride, and a mass/volume ratio of 0.1%-1% Dodecyl sulfate triethanolamine, volume/volume ratio of 0.1% to 1% ethyl phenyl polyethylene glycol (NP-40), mass/volume ratio of 0.01% to 2% sodium dodecyl sulfonate, The volume/volume ratio is one or more of the components such as 0.05% to 1% ethanol, but the present invention is not limited to this.
  • qPCR amplification reagents refer to reagents used for real-time fluorescent quantitative nucleic acid amplification detection. Those skilled in the art can understand that qPCR amplification reagents usually contain DNA polymerase, dNTP, PCR buffer, and the like. For example, when the detection target is RNA, reverse transcriptase may be further included. It is not difficult to understand that those skilled in the art can determine the composition and concentration of the PCR reaction reagents according to specific needs (such as virus type, content, etc.).
  • the "one-step method" mentioned in the present invention refers to a nucleic acid release and amplification technology (Extraction Free Nucleic Acid Release and Amplification Technology, EFNART) without sample extraction. It refers to direct sample nucleic acid amplification and detection by directly matching the sample nucleic acid release agent under strong alkaline conditions and a highly compatible amplification system without the need for nucleic acid extraction or purification of the sample.
  • EFNART extraction Free Nucleic Acid Release and Amplification Technology
  • the sample, the sample release agent, and the qPCR reaction solution in the preservation solution or pretreatment solution are mixed and directly amplified.
  • the mixing can be carried out in the usual proportions in the art.
  • the sample, the sample release agent, and the qPCR reaction solution in the preservation solution or the pretreatment solution may be about 5:5:40, about 10:10:30, or about 5:15:30 (v/ The ratio of v) exists, but the present invention is not limited to this.
  • the present invention provides a pretreatment solution for virus nucleic acid detection, and the pretreatment solution includes:
  • the pH of the pretreatment liquid is 6.5-8.0.
  • Tris-HCl may be present at a concentration of about 10 to about 200 mM, preferably at a concentration of about 80 to about 120 mM, and most preferably at a concentration of about 100 mM.
  • EDTA-2Na may be present at a concentration of about 8 to about 50 mM, preferably at a concentration of about 10 to about 15 mM, and most preferably at a concentration of about 10 mM.
  • sodium chloride may be present at a concentration of about 0.5% to about 2% (w/v), preferably at a concentration of about 0.8 to about 1% (w/v), and most preferably at a concentration of about 0.9% (w/v) The concentration of /v) exists.
  • the ribonuclease inhibitor may be present at a concentration of about 2U/mL to about 800U/mL, preferably at a concentration of about 10 to about 30U/mL, and most preferably at a concentration of 20U/mL exist.
  • the pH value is in the range of 6.5 to 8.0, preferably 7.0 to 8.0, and most preferably 7.5.
  • Antibiotics include but are not limited to Proclin antibiotics (such as Proclin 300 and Proclin 950) and NaN 3 .
  • Proclin 300 when used as an antibiotic, its concentration can be about 0.01% (v/v); for another example, when Proclin 950 is used as an antibiotic, its concentration can be about 0.04% (v/v) ; But the present invention is not limited to this.
  • the pretreatment solution has a concentration of Tris-HCl 100 mM, a concentration of EDTA-2Na 10 mM, a concentration of sodium chloride 0.9% (w/v), and a concentration of ribonuclease inhibitor: 20U/mL, the concentration of Proclin 950 is 0.04%(v/v);
  • the virus may be a DNA virus or an RNA virus.
  • the virus is an RNA virus.
  • the virus is a coronavirus (such as the 2019 novel coronavirus), respiratory syncytial virus, and enterovirus.
  • the present invention provides a use of a pretreatment solution in preparing a one-step nucleic acid amplification detection kit for virus detection.
  • the present invention provides a one-step-based viral nucleic acid detection kit, which includes the above-mentioned pretreatment solution.
  • the kit also includes a sample release agent and a qPCR amplification reagent.
  • FIG. 1 is a diagram showing the results of the "one-step” detection of a gradiently diluted 2019 novel coronavirus (2019-nCoV) nucleic acid of a sample pretreated by the pretreatment method of the present invention after being stored at room temperature for 72 hours.
  • 2019-nCoV 2019 novel coronavirus
  • Example 1 The present invention is used for the pretreatment and rapid detection of respiratory syncytial virus (RSV) oropharyngeal swab samples
  • the virus pretreatment solution of the invention (the concentration of Tris-HCl is 100 mM, the concentration of EDTA-2Na is 10 mM, the concentration of sodium chloride is 0.9% (w/v), The concentration of RNasin is 20U/mL, and the concentration of Proclin 950 is 0.04% (v/v)), compared with normal saline and commercial virus pretreatment solution.
  • the method of comparison is to dilute (1:9, v/v) pretreatment with clinically diagnosed positive respiratory syncytial virus (RSV) throat swab samples, and the pretreatment conditions are room temperature 25°C, respectively at 0 Hour/24 hours/48 hours and 72 hours for direct amplification of samples.
  • RSV positive respiratory syncytial virus
  • the detection efficiency of real-time qPCR (real-time qPCR) under room temperature pretreatment conditions is compared with the Ct value to evaluate the effects of different pretreatment solutions on viruses.
  • the above results show that for the virus pretreatment method of the present invention, pretreatment at room temperature for 24 hours can be well pretreated for serially diluted RSV samples, and can be directly used for EFNART detection, based on physiological saline.
  • the pretreatment method of the substrate After a long time pretreatment, the Ct value will be delayed, and the nucleic acid of the virus has been degraded to a certain extent, which affects the amplification efficiency.
  • the Hanks pretreatment solution and guanidine salt pretreatment solution commonly used in the market cannot provide effective virus pretreatment and PCR amplification detection methods in this scheme.
  • Example 2 The present invention is used for nucleic acid pretreatment and rapid detection of 2019 novel coronavirus (2019-nCoV) samples after purification
  • the virus pretreatment solution of the invention (the concentration of Tris-HCl is 100 mM, the concentration of EDTA-2Na is 10 mM, the concentration of sodium chloride is 0.9% (w/v), The concentration of RNasin is 20U/mL, and the concentration of Proclin 300 is 0.01% (v/v)), which is compared with normal saline and commercial virus pretreatment solution.
  • the comparison method is to dilute (1:9, v/v) pretreatment with the nucleic acid of the 2019 novel coronavirus (2019-nCoV) that is clinically diagnosed as positive. Hour/24 hours/48 hours and 72 hours for direct amplification of samples.
  • the detection efficiency of real-time qPCR (real-time qPCR) under room temperature pretreatment conditions is compared with the Ct value to evaluate the effects of different pretreatment solutions on viruses.
  • the results are shown in Table 3.
  • the nucleic acid of 2019 novel coronavirus (2019-nCoV) which is used in the virus pretreatment method of the present invention, is stored at room temperature for 72 hours, and the result of the “one-step method” detection is shown in Figure 1.
  • pretreatment at room temperature for 24 hours can perform well pretreatment for the gradient dilution of 2019-nCoV nucleic acid, and can be directly used for detection in EFNART, based on The pretreatment method of physiological saline matrix. After a long time pretreatment, the Ct value will be delayed, and the nucleic acid of the virus will be degraded to a certain extent, which affects the amplification efficiency. On the contrary, the Hanks pretreatment solution and guanidine salt pretreatment solution commonly used in the market cannot provide effective virus pretreatment and PCR amplification detection methods in this scheme.
  • Example 3 The present invention is used for the pretreatment and rapid detection of enterovirus universal (EV) throat swab samples
  • the virus pretreatment solution of the invention (the concentration of Tris-HCl is 100 mM, the concentration of EDTA-2Na is 10 mM, the concentration of sodium chloride is 0.9% (w/v), The concentration of SDS is 0.1%, and the concentration of Proclin 950 is 0.04% (v/v)), which is compared with normal saline and commercial virus pretreatment solution.
  • the method of comparison is to dilute (1:9, v/v) pretreatment with clinically diagnosed enterovirus universal (EV) throat swab samples.
  • the pretreatment conditions are room temperature 25°C, and the pretreatment time Direct amplification of samples at 0 hour/24 hours/48 hours and 72 hours is performed, and the detection efficiency of real-time qPCR under room temperature pretreatment conditions is compared with the Ct value to evaluate the pairing of different pretreatment solutions.
  • pretreatment at room temperature for 24 hours can be well pretreated for gradiently diluted EV samples, and can be directly used for detection in EFNART, based on physiological saline.
  • the pretreatment method of the substrate After a long time pretreatment, the Ct value will be delayed, and the nucleic acid of the virus has been degraded to a certain extent, which affects the amplification efficiency.
  • the Hanks pretreatment solution and guanidine salt pretreatment solution commonly used in the market cannot provide effective virus pretreatment and PCR amplification detection methods in this scheme.
  • Example 4 Pretreatment ability of the virus pretreatment solution of the present invention for RNA viruses
  • RNA viruses Compared with DNA viruses, RNA viruses have higher requirements for pretreatment and detection, and are more susceptible to environmental factors. Especially when the consumables used in the pretreatment process contain RNase, it has a vital impact on the detection of RNA viruses.
  • the concentration of Tris-HCl is 100 mM
  • the concentration of EDTA-2Na is 10 mM
  • the concentration of sodium chloride is 0.9% (w/v)
  • the concentration of Rnasin is 20 U/mL
  • Proclin The concentration of 950 is 0.04% (v/v)
  • the nucleic acid extracted from the 2019 novel coronavirus (2019-nCoV) sample pretreated with the virus pretreatment solution of the present invention is divided into two parts (A/ B) At the same time, prepare a nucleic acid (C) extracted from a sample of 2019 novel coronavirus (2019-nCoV) with the same concentration pretreated in normal saline, and add 0.25 ⁇ g/mL Rnase A, respectively to the A and C solutions. Three solutions containing 2019-nCoV nucleic acid were pretreated at room temperature (25°C) for 24 hours, and then the pretreatment solution of the present invention was pretreated and detected for RNA viruses using the EFNART "one-step method" technology.
  • Example 5 The virus pretreatment solution of the present invention is used for the pretreatment and rapid detection of hepatitis B virus (HBV) serum samples
  • the virus pretreatment solution of the invention (the concentration of Tris-HCl is 100 mM, the concentration of EDTA-2Na is 10 mM, and the concentration of sodium chloride is 100 mM). The concentration is 0.9% (w/v), the concentration of Rnasin is 20 U/mL, and the concentration of Proclin 950 is 0.04% (v/v)). Comparative analysis with physiological saline and commercial virus pretreatment solution. The method of comparison is to dilute (1:9, v/v) pretreatment with a serum sample of hepatitis B virus (HBV) that is clinically diagnosed as positive.
  • HBV hepatitis B virus
  • the pretreatment conditions are room temperature 25°C, respectively at 0 hours of pretreatment time /24 hours/48 hours and 72 hours to directly amplify the samples, and compare the detection efficiency of real-time qPCR (real-time qPCR) under room temperature pretreatment conditions by Ct value to evaluate the effects of different pretreatment solutions on DNA viruses The influence of pretreatment effect.
  • the initial design is for the virus preservation matrix used for the "one-step” amplification of RNA viruses, it can also be used for the preservation and one-step amplification of DNA viruses. Increase detection.
  • the relevant components in the present invention were adjusted and reduced, and then the sample was preserved for a pretreatment comparison test.
  • the separately optimized components included ribonuclease inhibition, antibiotics, and EDTA- 2Na, etc.
  • the adjusted concentrations of various components are shown in Table 7. Adjust the pretreatment solution configured with different concentrations to store the respiratory syncytial virus (RSV) at room temperature for 24 hours and then directly perform qPCR amplification detection, and compare the Ct value to real-time qPCR under room temperature pretreatment conditions ) To evaluate the effect of different pretreatment solutions on the effect of virus pretreatment.
  • RSV respiratory syncytial virus
  • the chemical component in the invention is an essential component, and it performs best in the pretreatment of RNA virus and the one-step RT-PCR detection in the concentration of the invention.

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Abstract

本发明属于病毒核酸检测领域。具体地，本发明提供了一种用于病毒核酸检测的预处理方法，所述方法包括将含有样本的预处理液与核酸释放剂和qPCR扩增试剂混合，其中，所述预处理液包括：Tris-HCl、EDTA-2Na、氯化钠、核糖核酸酶抑制剂和抗生素；其中，所述预处理液的pH为6.5～8.0。

Description

病毒核酸检测的预处理方法、预处理液、试剂盒及其用途 技术领域
本发明涉及病毒样本的核酸检测领域；特别地，涉及一种病毒样本预处理方法和预处理液，用于对DNA/RNA病毒进行预处理。
背景技术
现行传统的病毒预处理液主要有两种方式：基于Hank's基质的预处理液和基于胍盐的预处理液。Hank's液是病毒运送的常见的平衡盐溶液(Balanced Salt Solution，BSS)。受各种因素的影响，Hank's液保存呼吸道病毒(如流感病毒、新冠病毒等)只能保存数个小时，数个小时之后病毒形态就会受到影响，进而会影响到病毒的核酸检测效率，如果时间再延长一些，那么还很容易滋生细菌、真菌，导致Hank's液pH值下降，加速病毒降解，所以很难用于病毒的长期保存与核酸检测。而基于胍盐的病毒预处理液中的盐酸胍或者Rnasin的浓度相对而言较高(3-5M)，高浓度的胍盐适用于常温下的病毒灭活。但是由于胍盐浓度太高，对于诸如磁珠法的核酸提取或纯化有很大的抑制作用，需要多次洗涤或者用于离心柱的方式才可进行。
样本免提取的核酸释放及扩增技术(Extraction Free Nucleic Acid Release and Amplification Technology，EFNART),简称“一步法”技术，是指在不需要对样本进行核酸提取或纯化的情况下，直接搭配强碱性质下的样本核酸释放剂和高兼容性的扩增体系，进行直接的样本核酸扩增检测。这样的“一步法”操作将大大节省病毒核酸检测尤其是RNA病毒的检测时间，预计相比较传统的核酸提取后的扩增方法，该发明可以节省60％以上的时间，检测效率提升50％。
在发生重大疫情(例如，2019-2020年发生的新冠2019-nCoV疫情)的时候，更加倾向于一步法检测，可以节省检测时间，提高效率，会对整个疫情的 攻克带来巨大的贡献。
但是，现行常见的病毒预处理液，例如Hank's和基于胍盐的预处理液均不能很好的用于后续的一步法检测，从而很难用于病毒样本的快速检测和筛查的应用场景中。
因此，本领域需求一种能够很好适配于基于一步法的病毒核酸检测的样本预处理液。
发明内容
有鉴于此，第一方面，本发明提供一种用于病毒核酸检测的预处理方法，所述方法包括将存放在预处理液中的样本与核酸释放剂和qPCR扩增试剂混合，
其中，所述预处理液包括：Tris-HCl、EDTA-2Na、氯化钠、核糖核酸酶抑制剂和抗生素；
其中，所述预处理液的pH为6.5～8.0。
在本发明中，Tris-HCl可以约10～约200mM的浓度存在，优选以约80～约120mM的浓度存在，最优选以约100mM的浓度存在。
在本发明中，EDTA-2Na可以约8～约50mM的浓度存在，优选以约10～约15mM的浓度存在，最优选以约10mM的浓度存在。
在本发明中，氯化钠可以约0.5％～约2％(w/v)的浓度存在，优选以约0.8～约1％(w/v)的浓度存在，最优选以约0.9％(w/v)的浓度存在。
在本发明中，“核糖核酸酶抑制剂”是指能够对核糖核酸酶进行抑制，使其失活的化学物质。包括但不限于焦碳酸二乙酯(DEPC)、RNA酶的蛋白质抑制剂(RNasin)、氧钒核糖核苷复合物、SDS等。
在本发明中，核糖核酸酶抑制剂(RNase抑制剂)可以约2U/mL～约800U/mL的浓度存在，例如，为约40U/mL、约50U/mL、约100U/mL；优选地，以约10～约30U/mL的浓度存在；最优选地，以20U/mL的浓度存在。
在本发明中，pH值的范围为6.5～8.0，优选为7.0～8.0，最优选为7.5。
抗生素包括但不限于Proclin类抗生素(如Proclin 300和Proclin 950)和NaN 3。
例如，在使用Proclin 300作为抗生素的情况下，其浓度可以为约0.01％(v/v)；又例如，在使用Proclin 950作为抗生素的情况下，其浓度可以为约0.04％(v/v)；但本发明不限于此。
在一个具体的实施方案中，所述预处理液为Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、核糖核酸酶抑制剂的浓度为20U/mL，Proclin 950的浓度为0.04％(v/v)；
并将上述预处理液调至pH值为7.5。
如本文所使用的，“样本”是指可能含有病毒的样本。所述样本可来源于人或动物的血液、粪便、尿液、口腔上皮细胞、脱落细胞、口腔拭子、咽拭子等。
本发明中提及的“预处理”是指对样本进行检测之前的处理，尤其指在对样本进行(基于一步法的)核酸检测前的处理。
本发明中提及的“预处理液”是指对病毒样本进行预处理的液体。
在本发明中，病毒可为DNA病毒或RNA病毒。
在一个优选地实施方案中，所述病毒为RNA病毒。
在更优选地实施方案中，所述病毒为冠状病毒(如2019新型冠状病毒)、呼吸道合胞病毒、肠道病毒。
使用本发明的预处理方法配置的核酸检测反应液，能够直接进行qPCR，无需提取或纯化过程，提高检测效率，缩短检测时间。该发明中采用的抗生素和核糖核酸酶抑制剂可以用于DNA病毒和RNA病毒的预处理，可以避免在常温状态下因为细菌等多种微生物的生长影响病毒保存的活性。RNA病毒由于随处可在的RNA酶的原因，更容易降解，使用本发明的方法同时避免医学采样过程中(口腔上皮细胞、脱落细胞、咽拭子等多种样本类型)由于样本本身或者采样耗材本身带来的对RNA的破坏，非常有利于RNA病毒的长期保存和检测。
需要说明的是，在本发明的预处理液处理的样品不进行存放而是直接(即处理后0hr)进行EFNART检测的情况下，如下文实施例中所证实的，本发明预处理液的组分具有增强RT-PCT的作用。
在第二方面，本发明提供了一种对样本中病毒核酸进行检测的方法，包括使用前述方法配置的反应液直接进行qPCR扩增而检测。
样本释放剂是指能够释放样本中核酸的化学试剂。例如强酸性或强碱性的化学试剂。示例性的样本释放剂可以是包括0.01～0.5mmol/L莎梵婷(surfactin)、100～200mmol/L氯化钾、50～200mmol/L氯化锂、质量/体积比为0.1％～1％十二烷基硫酸三乙醇胺、体积/体积比为0.1％～1％乙基苯基聚乙二醇(NP-40)、质量/体积比为0.01％～2％十二烷基磺酸钠、体积/体积比为0.05％～1％乙醇等组分中的一种或几种，但本发明不限于此。
qPCR扩增试剂是指用于实时荧光定量核酸扩增检测的试剂。本领域技术人员能够理解qPCR扩增试剂通常含有DNA聚合酶、dNTP、PCR缓冲液等。例如当检测对象为RNA时，还可进一步包括逆转录酶。不难理解，本领域技术人员可以根据具体需要(例如病毒的种类，含量等)，确定PCR反应试剂的成分和浓度。
本发明中提及的“一步法”是指样本免提取的核酸释放及扩增技术(Extraction Free Nucleic Acid Release and Amplification Technology,EFNART)。其是指在不需要对样本进行核酸提取或纯化的情况下，直接搭配强碱性质下的样本核酸释放剂和高兼容性的扩增体系，进行直接的样本核酸扩增检测。
在基于一步法对样本中的病毒核酸进行检测时，将存在于保存液或预处理液中的样本、样本释放剂和qPCR反应液混合后直接扩增。混合可以本领域中通常比例进行。在示例性的实施方案中，在于保存液或预处理液中的样本、样本释放剂和qPCR反应液可以约5:5:40、约10:10:30或者约5:15:30(v/v)的比例存在，但本发明不限于此。
第三方面，本发明提供一种用于病毒核酸检测的预处理液，所述预处理液包括：
Tris-HCl、EDTA-2Na、氯化钠、核糖核酸酶抑制剂和抗生素；
其中，所述预处理液的pH为6.5～8.0。
在本发明中，Tris-HCl可以约10～约200mM的浓度存在，优选以约80～约120mM的浓度存在，最优选以约100mM的浓度存在。
在本发明中，EDTA-2Na可以约8～约50mM的浓度存在，优选以约10～约15mM的浓度存在，最优选以约10mM的浓度存在。
在本发明中，氯化钠可以约0.5％～约2％(w/v)的浓度存在，优选以约0.8～约1％(w/v)的浓度存在，最优选以约0.9％(w/v)的浓度存在。
在本发明中，核糖核酸酶抑制剂(RNase抑制剂)可以约2U/mL～约800U/mL的浓度存在，优选以约10～约30U/mL的浓度存在，最优选以20U/mL的浓度存在。
在本发明中，pH值的范围为6.5～8.0，优选为7.0～8.0，最优选为7.5。
抗生素包括但不限于Proclin类抗生素(如Proclin 300和Proclin 950)和NaN 3。
例如，在使用Proclin 300作为抗生素的情况下，其浓度可以为约0.01％(v/v)；又例如，在使用Proclin 950作为抗生素的情况下，其浓度可以为约0.04％(v/v)；但本发明不限于此。
在一个具体的实施方案中，所述预处理液为Tris-HCl的浓度100mM、EDTA-2Na的浓度10mM、氯化钠的浓度为0.9％(w/v)、核糖核酸酶抑制剂的浓度为20U/mL，Proclin 950的浓度为0.04％(v/v)；
并将上述预处理液调至pH值为7.5。
在一些具体实施方案中，病毒可为DNA病毒或RNA病毒。
在一个优选地实施方案中，所述病毒为RNA病毒。
在更优选地实施方案中，所述病毒为冠状病毒(如2019新型冠状病毒)、呼吸道合胞病毒、肠道病毒。
第四方面，本发明提供一种预处理液在制备用于检测病毒的一步法核酸扩增检测试剂盒中的用途。
第五方面，本发明提供一种基于一步法的病毒核酸检测的试剂盒，所述试剂盒包括上述的预处理液。
进一步地，所述试剂盒还包括样本释放剂和qPCR扩增试剂。
附图说明
图1为本发明预处理方法预处理的样品的梯度稀释的2019新型冠状病毒(2019-nCoV)核酸室温保存72小时之后“一步法”检测的结果图。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1 本发明用于呼吸道合胞病毒(RSV)口咽拭子样本预处理和快速检测
为了测评该发明中的病毒预处理液，将该发明的病毒预处理液(Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、RNasin浓度为20U/mL，Proclin 950的浓度为0.04％(v/v))与生理盐水以及商品化的病毒预处理液进行对比分析。对比的方法为用临床诊断为阳性的呼吸道合胞病毒(RSV)咽拭子样本进行稀释(1:9，v/v)预处理，预处理条件为室温25℃，分别在预处理时间的0小时/24小时/48小时和72小时进行样本的直接扩增，通过Ct值对比在室温预处理条件下的实时荧光定量PCR(real-time qPCR)的检测效率，来评价不同预处理液对病毒预处理效果的影响。qPCR扩增检测的方式为上述的EFNART“一步法”技术，利用带有样本的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。
实时荧光定量PCR(Real-time qPCR)扩增检测程序如表1所示，结果如表2所示。
表1
Figure PCTCN2020090052-appb-000001
表2
Figure PCTCN2020090052-appb-000002
上述结果表明，对于该发明中的病毒预处理方式，在室温下预处理24小时，对于梯度稀释的RSV样本能够很好的进行预处理，并且可以直接用于EFNART的方式进行检测，基于生理盐水基质的预处理方式，长时间预处理后，Ct值会拖后，病毒的核酸得到了一定的降解，影响了扩增效率。反而市面上常用的Hanks预处理液和胍盐预处理液在该方案中均不能提供有效的病毒预处理和PCR扩增检测的方式。
实施例2 本发明用于2019新型冠状病毒(2019-nCoV)样本纯化后核酸预处理和快速检测
为了测评该发明中的病毒预处理液，将该发明的病毒预处理液(Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、RNasin的浓度为20U/mL，Proclin 300的浓度为0.01％(v/v))与生理盐水以 及商品化的病毒预处理液进行对比分析。对比的方法为用临床诊断为阳性的2019新型冠状病毒(2019-nCoV)的核酸进行稀释(1:9，v/v)预处理，预处理条件为室温25℃，分别在预处理时间的0小时/24小时/48小时和72小时进行样本的直接扩增，通过Ct值对比在室温预处理条件下的实时荧光定量PCR(real-time qPCR)的检测效率，来评价不同预处理液对病毒预处理效果的影响。qPCR扩增检测的方式为上述的EFNART“一步法”技术，利用带有核酸的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。结果如表3所示。用于该发明中的病毒预处理方法梯度稀释的2019新型冠状病毒(2019-nCoV)核酸室温保存72小时，“一步法”检测的结果如图1所示。
表3
Figure PCTCN2020090052-appb-000003
上述结果表明，对于该发明中的病毒预处理方式，在室温下预处理24小时，对于梯度稀释的2019-nCoV核酸能够很好的进行预处理，并且可以直接用于EFNART的方式进行检测，基于生理盐水基质的预处理方式，长时间预处理后，Ct值会拖后，病毒的核酸得到了一定的降解，影响了扩增效率。反而市面上常用的Hanks预处理液和胍盐预处理液在该方案中均不能提供有效的病毒预处理和PCR扩增检测的方式。
实施例3 本发明用于肠道病毒通用性(EV)咽拭子样本预处理和快速检 测
为了测评该发明中的病毒预处理液，将该发明的病毒预处理液(Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、SDS的浓度为0.1％，Proclin 950的浓度为0.04％(v/v))与生理盐水以及商品化的病毒预处理液进行对比分析。对比的方法为用临床诊断为阳性的肠道病毒通用型(EV)咽拭子样本进行稀释(1:9，v/v)预处理，预处理条件为室温25℃，分别在预处理时间的0小时/24小时/48小时和72小时进行样本的直接扩增，通过Ct值对比在室温预处理条件下的实时荧光定量PCR(real-time qPCR)的检测效率，来评价不同预处理液对病毒预处理效果的影响。qPCR扩增检测的方式为上述的EFNART“一步法”技术，利用带有样本的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。结果如表4所示：
表4
Figure PCTCN2020090052-appb-000004
上述结果表明，对于该发明中的病毒预处理方式，在室温下预处理24小时，对于梯度稀释的EV样本能够很好的进行预处理，并且可以直接用于EFNART的方式进行检测，基于生理盐水基质的预处理方式，长时间预处理后，Ct值会拖后，病毒的核酸得到了一定的降解，影响了扩增效率。反而市面上常用的Hanks预处理液和胍盐预处理液在该方案中均不能提供有效的病毒预处理和PCR扩增检测的方式。
实施例4 本发明的病毒预处理液对于RNA病毒的预处理能力
相对于DNA病毒，由于RNA病毒的预处理和检测有更高的要求，更容易受到环境因素的影响。尤其是预处理过程中使用的耗材中含有RNA酶的存在的情况下，对RNA病毒的检测有着至关重要的影响。为了测评该发明中的病毒预处理液(Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、Rnasin的浓度为20U/mL，Proclin 950的浓度为0.04％(v/v))对RNA病毒的预处理效果，将该发明的病毒预处理液预处理的2019新型冠状病毒(2019-nCoV)样本提取的核酸分成两份(A/B)，同时制备一份近在生理盐水中预处理的同等浓度的2019新型冠状病毒(2019-nCoV)样本提取的核酸(C)，分别往A和C溶液中加入0.25μg/mL Rnase A,将三种含有2019-nCoV核酸的溶液进行室温(25℃)预处理24小时，然后采用EFNART“一步法”技术对该发明中的预处理液进行对RNA病毒的预处理和检测。检测方式如下：利用带有核酸的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。结果如表5所示。
表5
Figure PCTCN2020090052-appb-000005
通过上述实验证明，以预处理的核酸样品D为参照的情况下，该发明中加入0.25μg/mL Rnase A对2019-nCoV的核酸检测效果无影响，该发明中的有效组分能够消化RNA酶，减少RNA酶对实验检测的影响，能够保证对RNA病毒直接检测的有效率。以预处理的核酸样品C来看，实验条件中加入的0.25μg/mL Rnase A能够消化降解实验中的RNA，因此对RNA病毒的直接检测造成很大的影响，引起漏检的风险。
实施例5 本发明的病毒预处理液用于乙型肝炎病毒(HBV)血清样本预处理和快速检测
为了测评该发明中的病毒预处理液在DNA病毒保存和扩增检测的作用，将该发明的病毒预处理液(Tris-HCl的浓度为100mM、EDTA-2Na的浓度为10mM、氯化钠的浓度为0.9％(w/v)、Rnasin的浓度为20U/mL，Proclin 950的浓度为0.04％(v/v))与生理盐水以及商品化的病毒预处理液进行对比分析。对比的方法为用临床诊断为阳性的乙型肝炎病毒(HBV)的血清样本进行稀释(1:9，v/v)预处理，预处理条件为室温25℃，分别在预处理时间的0小时/24小时/48小时和72小时进行样本的直接扩增，通过Ct值对比在室温预处理条件下的实时荧光定量PCR(real-time qPCR)的检测效率，来评价不同预处理液对DNA病毒预处理效果的影响。qPCR扩增检测的方式为上述的EFNART“一步法”技术，利用带有DNA病毒的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。结果如表6所示。
表6
Figure PCTCN2020090052-appb-000006
上述结果表明，对于该发明中的病毒预处理方式，虽然最开始的设计针对 于RNA病毒用于“一步法”扩增的病毒保存基质，但同时也可以用于DNA病毒的保存和一步法扩增检测。
实施例6 本发明的病毒预处理液的各种组分的有效性的验证
为了验证本发明中的各种组分的有效性，将该发明中的相关组分进行调整降低后对样本保存进行预处理对比试验，单独优化组分分别包括核糖核酸酶抑制跟抗生素、EDTA-2Na等，调整的各种组分的浓度如表7。调整不同浓度配置成的预处理液对呼吸道合胞病毒(RSV)进行室温保存24小时后直接进行qPCR扩增检测，通过Ct值对比在室温预处理条件下的实时荧光定量PCR(real-time qPCR)的检测效率，来评价不同预处理液对病毒预处理效果的影响。qPCR扩增检测的方式为上述的EFNART“一步法”技术，利用带有样本的预处理液：核酸释放剂：PCR扩增试剂＝10:10:30的方式，直接在PCR扩增管中进行real-time qPCR扩增检测。
表7
Figure PCTCN2020090052-appb-000007
通过上述实验，该发明中的化学组分是必需成分，并且在该发明中的浓度中对RNA病毒的预处理和一步法RT-PCR检测中表现最优。

Claims (12)

  1. 一种用于病毒核酸检测的预处理方法，所述方法包括将存放在预处理液中的样本与核酸释放剂和qPCR扩增试剂混合，
    其中，所述预处理液包括：Tris-HCl、EDTA-2Na、氯化钠、核糖核酸酶抑制剂和抗生素；
    其中，所述预处理液的pH为6.5～8.0。
  2. 根据权利要求1所述的方法，其中，在所述预处理液中，Tris-HCl的浓度为10～200mM，EDTA-2Na的浓度为8～50mM，氯化钠的浓度为0.5％～2％(w/v)，核糖核酸酶抑制剂的浓度为2U/mL～800U/mL，并且抗生素的浓度为0.005～0.05％。
  3. 根据权利要求1所述的方法，其中，在所述预处理液中，Tris-HCl的浓度为80～120mM、EDTA-2Na的浓度为10～15mM、氯化钠的浓度为0.8～1％(w/v)、核糖核酸酶抑制剂的浓度为10～30U/mL。
  4. 根据权利要求1所述的方法，其中，该方法所制备的反应液可直接进行qPCR扩增检测。
  5. 根据权利要求1～4中任一项所述的方法，其中，所述病毒为DNA病毒和RNA病毒，优选为RNA病毒，更优选为冠状病毒，如2019新型冠状病毒。
  6. 一种用于病毒核酸检测的预处理液，所述预处理液包括：Tris-HCl、EDTA-2Na、氯化钠、核糖核酸酶抑制剂和抗生素；
    其中，所述预处理液的pH为6.5～8.0。
  7. 根据权利要求6所述的预处理液，其中，在所述预处理液中，Tris-HCl的浓度为10～200mM，EDTA-2Na的浓度为8～50mM，氯化钠的浓度为0.5％～2％(w/v)，核糖核酸酶抑制剂的浓度为2U/mL～800U/mL，并且抗生素的浓度为0.005～0.05％。
  8. 根据权利要求6所述的预处理液，其中，在所述预处理液中，Tris-HCl的浓度为80～120mM、EDTA-2Na的浓度为10～15mM、氯化钠的浓度为0.8～1％ (w/v)、核糖核酸酶抑制剂的浓度为10～30U/mL。
  9. 根据权利要求6～8中任一项所述的预处理液，其中，所述病毒为DNA病毒和RNA病毒。
  10. 权利要求6～9中任一项所述的预处理液在制备用于检测病毒的一步法核酸扩增检测试剂盒中的用途。
  11. 基于一步法的病毒核酸检测的试剂盒，所述试剂盒包括权利要求6～9中任一项所定义的预处理液。
  12. 根据权利要求11所述的试剂盒，其中，所述试剂盒进一步包括样本释放剂和qPCR扩增试剂。
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