WO2021248779A1 - 一种拭子样本核酸释放剂及其应用 - Google Patents

一种拭子样本核酸释放剂及其应用 Download PDF

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WO2021248779A1
WO2021248779A1 PCT/CN2020/124134 CN2020124134W WO2021248779A1 WO 2021248779 A1 WO2021248779 A1 WO 2021248779A1 CN 2020124134 W CN2020124134 W CN 2020124134W WO 2021248779 A1 WO2021248779 A1 WO 2021248779A1
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nucleic acid
sample
release agent
buffer
swab
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PCT/CN2020/124134
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English (en)
French (fr)
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何蕴韶
蒋析文
夏乔
董志强
廖丽丽
李汉荣
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广州达安基因股份有限公司
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Priority to US17/284,476 priority Critical patent/US11753672B2/en
Priority to EP20866968.9A priority patent/EP3943612A4/en
Publication of WO2021248779A1 publication Critical patent/WO2021248779A1/zh

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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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  • the invention belongs to the technical field of pathogen molecular detection. More specifically, it relates to a swab sample nucleic acid release agent with simple operation and rapid release and its application.
  • Nucleic acid molecular detection has incomparable advantages in the detection and screening of pathogens and has a wide range of applications. For example, in the recent outbreak of the new coronavirus, nucleic acid testing has played a key role in the screening and testing of infected and asymptomatic people, and is of great significance to epidemic control.
  • swab samples are mainly used, including nasal swabs, throat swabs, oral swabs, anal swabs, etc., because they are relatively collected, transported, and stored. Convenient, and is widely adopted.
  • nucleic acid extraction is an indispensable part of nucleic acid detection.
  • Nucleic acid extraction and purification generally go through the steps of lysis-washing-elution of the sample.
  • the main nucleic acid extraction reagents on the market currently include column extraction and magnetic bead methods.
  • the nucleic acid extracted by the column extraction method and the magnetic bead method has the advantages of high purity, but at the same time there are the disadvantages that the operation is cumbersome and the corresponding extraction equipment is required, which leads to the high cost. It is very unsuitable for swab samples that are inherently small and difficult to store and transport.
  • the storage of swab samples in the market is basically based on physiological saline or sample preservation solution, and a certain amount of samples need to be taken for nucleic acid extraction during use.
  • the collected swab samples generally require high temperature inactivation. When high temperature inactivation is performed, more aerosol pollution is likely to occur, which makes laboratories and experimental operators have a higher risk of contamination.
  • Patent 201811598212.9 provides an elution lysis solution for eluting and lysing the cells on the swab to release nucleic acid.
  • the method of using the lysis solution still requires high temperature processing of the sample, and two steps are required: the swab is first Sub-samples are immersed in the eluted lysate, vortexed to mix, and incubated at 37°C for 60 minutes; then after removing the swab again, the centrifuge tube after taking out the swab is bathed in a water bath at 65°C for 5 minutes at 95°C Take a water bath for 10 minutes, and take the supernatant as the test solution; it can be seen that the method is not as simple as it claims, and it takes a long time, and high temperature treatment is easy to damage nucleic acid to a certain extent, lose the amount of nucleic acid, and easily produce the above-mentioned aerosol pollution problem.
  • the current nucleic acid extraction of swab samples has at least the following disadvantages: (1) high temperature inactivation of the sample is required, there is a risk of contamination to the operator and the laboratory, and the amount of nucleic acid will be lost; (2) the sample needs to be For additional nucleic acid extraction, the operation process is more complicated, and the time from collecting samples to the results is increased clinically; (3) There are certain professional skills requirements for test personnel, and the quality of test results is very dependent on the operation process of test personnel. Big relationship.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned defects and deficiencies of the prior art, provide a simple, efficient, and rapid nucleic acid extraction reagent and extraction method for swab samples, and a direct lysis method to release nucleic acid in the sample.
  • the sample release agent of the present invention Under the action of the sample release agent of the present invention, the cell membrane is quickly destroyed, so that the nucleic acid is released, and the released nucleic acid can be added to the PCR system for amplification and detection.
  • the sample release agent of the present invention can be used for the nucleic acid extraction of nasopharyngeal swab samples, and the whole process is extremely simple and fast, without any opening operation in the middle, and no high temperature treatment, which can release and obtain the nucleic acid in the sample most efficiently, It will not affect the subsequent PCR amplification, and will not cause any risk of contamination and infection.
  • the purpose of the present invention is to provide a nucleic acid release agent for swab samples.
  • Another object of the present invention is to provide the application of the nucleic acid release agent for the swab sample.
  • a nucleic acid release agent for swab samples including the following components:
  • the metal ion chelating agent includes but not limited to one or more of EDTA, EGTA or citric acid.
  • the buffer includes but is not limited to Tris buffer, sodium citrate buffer, sodium phosphate buffer or sodium acetate buffer.
  • the polar organic solvent includes but is not limited to one or more of DMSO, hexamethylphosphoramide, and tetramethylethylenediamine.
  • the surfactant includes but is not limited to one or more of Triton X-100, NP-40, Tween-20 or SDS.
  • the nuclease inhibitor includes, but is not limited to, guanidine hydrochloride, guanidine isothiocyanate, or 4,4'-carbonyl bis(2-(1-naphthylamide)benzoic acid).
  • the concentration of the metal ion chelating agent is 5 mol/L, and the concentration of the buffer is 50 mmol/L and pH 8.5.
  • the concentration of the polar organic solvent is 25%.
  • the concentration of the surfactant is 2%.
  • the concentration of the nuclease inhibitor is 0.3 mol/L.
  • the method of using the sample release agent of the present invention is as follows: put the swab into a container containing the sample release agent, and at room temperature, turn the container upside down 5-10 times or mix well for 20-40 seconds, Let stand for 5-20 minutes (preferably 5-10 minutes), turn it upside down again for 5-10 times or mix for 20-40 seconds. After instant centrifugation, the supernatant is the released nucleic acid solution, and the obtained nucleic acid solution can be used for PCR Amplification.
  • the swab sample is stored, transported, and verified and released in the sample release agent. There is no need to open the cover throughout the whole process. It is safe, efficient, and extremely simple to use and has a wide range of applications.
  • sample release agent in the preparation of a swab sample nucleic acid release kit, and the developed swab sample nucleic acid release kit containing the sample release agent should fall within the protection scope of the present invention.
  • the sample release agent developed in the present invention can integrate the three steps of sampling, preservation, inactivation, and nucleic acid extraction into one step. After sampling, it can be directly put into the sampling tube containing the sample release agent for storage and transportation; when in use, turn the sampling tube upside down and mix well. After the sample and the sample release agent are fully mixed, let stand at room temperature for 15 minutes. When the sample is inactivated, the nucleic acid of the sample is released. The entire operation process does not require heating and halfway opening of the lid, which effectively avoids the contamination of experimental operators and laboratories, and simplifies the nucleic acid extraction operation to the greatest extent.
  • sample preservation function After the sample nucleic acid is released, it can protect the nucleic acid from degradation for a long time, and the nucleic acid can be stored for 72 hours without degradation under normal temperature conditions;
  • the present invention is a good supplement and alternative solution.
  • the sample release agent of the present invention can be used for sample collection-storage-transport-inactivation-nucleic acid extraction. It has obvious advantages such as speed, safety, stability, high efficiency and low cost.
  • the "inactivation” mentioned in this article refers to: the polar organic solvents and surfactants contained in the release agent can theoretically rupture cells and denature proteins to a certain extent, so that the virus inactivates and does not have the ability to infect. .
  • Figure 1 is a computer-based fluorescence quantitative PCR amplification diagram of the new crown clinical samples 1 and 2 extracted from the sample release agent of the present invention and the release agent of a commercially available company.
  • Figure 2 is a computer-based fluorescence quantitative PCR amplification diagram of the new crown clinical samples 1 and 2 extracted from the sample release agent of the present invention and the release agent of a commercial company.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • a nucleic acid release agent for swab samples including the following components:
  • Metal ion chelating agent 5mol/L EDTA
  • the method of using the above sample release agent is: put the collected nasopharyngeal swab into the sample collection tube containing the sample release agent, and at room temperature, turn the sampling tube containing the sample upside down and mix for 30 seconds. Let it stand for 5 minutes and turn it upside down again for 30 seconds. After instant centrifugation, the sample nucleic acid is released into the sample release agent, and the supernatant is the released nucleic acid solution.
  • the obtained nucleic acid solution can be directly pipetted into the PCR reaction solution, and then amplified on the PCR amplification machine.
  • a nucleic acid release agent for swab samples including the following components:
  • Metal ion chelating agent 10mol/L EGTA;
  • Nuclease inhibitor 0.5mol/L guanidine isothiocyanate.
  • a nucleic acid release agent for swab samples including the following components:
  • Metal ion chelating agent 1mol/L citric acid
  • Polar organic solvent 40% hexamethylphosphoramide
  • Nuclease inhibitor 1mol/L 4,4'-carbonyl bis(2-(1-naphthylamide)benzoic acid).
  • a nucleic acid release agent for swab samples including the following components:
  • Metal ion chelating agent 20mol/L EDTA
  • Polar organic solvent 5% tetramethylethylenediamine
  • Nuclease inhibitor 1mol/L guanidine hydrochloride.
  • a nucleic acid release agent for swab samples including the following components:
  • Metal ion chelating agent 1mol/L EDTA
  • Nuclease inhibitor 0.1mol/L guanidine hydrochloride.
  • the clinical positive samples 1 and 2 of the novel coronavirus collected in Guangzhou from January to March 2020 were respectively extracted.
  • the obtained nucleic acid solution is used as a template, directly pipetted into the PCR reaction solution, and amplified by the PCR amplification machine.
  • a release agent purchased from a certain company was used as a control. Take clinical positive samples of swabs sampled in parallel at the same time for nucleic acid extraction according to the manufacturer's instructions, and take the extracted nucleic acid for PCR detection.
  • the PCR amplification kit used in the nucleic acid PCR detection is the new coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) of Daan Gene Co., Ltd. of Sun Yat-sen University (National Machinery Approval 20203400063).
  • Amplification system Template amount total capacity 20 5 25
  • Clinical samples Clinical positive samples 3 and 4 of the novel coronavirus collected in Guangzhou from January to March 2020 (provided by Yunkang Clinical Laboratory Center)
  • Example 1 the novel coronavirus clinical samples 3 and 4 were stored using the sample release agent of the present invention. They were placed at room temperature for 3 days, 4°C for 7 days, and -20°C for 15 days, and then fluorescent quantitative PCR was performed.
  • Amplification, PCR amplification kit used for PCR detection is Sun Yat-Sen University Daan Gene Co., Ltd. new coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) (National Machinery Approval 20203400063).
  • the Ct value data is shown in Table 4 to Table 6.
  • the nucleic acid is stored stably, indicating that the sample release agent of the present invention has a very good storage effect on nucleic acid samples regardless of whether it is a low temperature of -20°C, 4°C, or normal temperature.
  • Tris buffer pH8.5, 50mmol/L Tris buffer
  • Polar organic solvent 25% DMSO
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • Metal ion chelating agent 5mol/L EDTA;
  • Tris buffer pH8.5, 50mmol/L Tris buffer
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • Metal ion chelating agent 5mol/L EDTA;
  • Tris buffer pH8.5, 50mmol/L Tris buffer
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • Tris buffer pH8.5, 50mmol/L Tris buffer
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • Metal ion chelating agent 5mol/L EDTA;
  • Polar organic solvent 25% DMSO
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • Metal ion chelating agent 5mol/L EDTA;
  • Tris buffer pH8.5, 50mmol/L Tris buffer
  • Polar organic solvent 25% DMSO
  • Nuclease inhibitor 0.3mol/L guanidine hydrochloride.
  • the novel coronavirus clinical sample 3 is treated with the sample release agent of the present invention, and then subjected to fluorescence quantitative PCR amplification.
  • the PCR amplification kit used for the PCR detection is Sun Yat-sen University Daan Gene Co., Ltd. New Coronavirus Virus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (National Machinery Standard Note 20203400063).
  • nucleic acid samples extracted using the 1-6 release agents of the experimental group and the comparison group were subjected to fluorescence quantitative PCR amplification after being placed at room temperature for 3 days.
  • the Ct value data is shown in Table 8.

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Abstract

本发明公开了一种拭子样本核酸释放剂及其应用。所述样本释放剂包括1-20mol/L的金属离子螯合剂、pH 6-9浓度5-80mmol/L的缓冲液、5％-40％（v/v）的极性有机溶剂、0.2％-10％（v/v）的表面活性剂、0.1-1mol/L的核酸酶抑制剂。该样本释放剂可以集取样保存-灭活-核酸提取三步为一步，从取样到PCR检测之间整个操作过程不用加热及中途开盖，操作简单。

Description

一种拭子样本核酸释放剂及其应用 技术领域
本发明属于病原体分子检测技术领域。更具体地，涉及一种操作简单、快速释放的拭子样本核酸释放剂及其应用。
背景技术
核酸分子检测在病原体的检测筛查方面具有不可比拟的优势，应用广泛。如近期爆发的新型冠状病毒疫情，核酸检测在感染人群以及无症状人群的筛查检测工作中起到了关键性的作用，对疫情控制意义重大。目前应用于病毒等病原体检测、遗传病筛查等检测工作都主要使用的是拭子样本，包括鼻拭子、咽拭子、口腔拭子、肛门拭子等，因其采集、运输、保存较为方便，而被大量采用。但是使用拭子收集的细胞量非常有限，而且细胞的生长和保存的条件都极为严苛，经过运输、常温储存，对拭子中的细胞造成大量的损伤，导致下游实验很容易造成假阳性或假阴性。
同时核酸提取是核酸检测中必不可少的环节，核酸提取与纯化一般会经过样本的裂解-洗涤-洗脱等步骤，目前市面上主要的核酸提取试剂有柱提法和磁珠法。柱提法和磁珠法提取的核酸具有纯度高的优点，但同时存在操作繁琐及需要用到相应的提取仪器导致成本较高的缺陷。对于样本量本就较少且保存运输困难的拭子样本而言非常不适用。目前市场上对于拭子类样本的保存基本上都是基于生理盐水或样本保存液，在使用时需要取一定量的样本进行核酸提取。对于采集到的拭子类样本一般需要高温灭活，在进行高温灭活时，容易产生较多的气溶胶污染，从而使实验室和实验操作人员存在较高的被污染风险。
专利201811598212.9提供了一种用于将拭子上的细胞洗脱、裂解释放核酸的洗脱裂解液，但是该裂解液的使用方法中仍需对样本进行高温处理，而且需要两步：先将拭子样本浸泡于所述洗脱裂解液中，涡旋混匀，37℃下孵育60min；然后再次开盖取出拭子后，将取出拭子后的离心管在65℃下水浴5min，95℃下水浴10min，取上清液作为待检测液；可见方法并未如其宣称的那样简便，且耗时长，而且高温处理易一定程度上破坏核酸、损失核酸量，且易产生上文所述的气溶胶污染问题。
总之，目前的拭子类样本的核酸提取至少存在以下几类缺点：(1)需要高温灭活样本，存在对操作人员和实验室的污染风险，且会损失核酸量；(2)需要对样本进行额外的核酸提取，操作过程较复杂，临床上增加了从收集样本到出结果的时间；(3)对试验人员有一定的专业技能要求，检测结果的好坏与试验人员的操作过程有很大关系。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足，提供一种简单、高效、快速的拭子类样本核酸提取试剂和提取方法，是一种直接裂解法释放样本中的核酸。在本发明样本释放剂的作用下，细胞膜被迅速破坏，使核酸释放出来，释放出来的核酸即可加入到PCR体系中扩增检测。本发明样本释放剂可用于鼻咽拭子样本的核酸提取，且整个过程极其简便、快速，中间无需任何一次开盖操作，不需任何高温处理，既能最高效的释放获取样本中的核酸，不影响后续PCR扩增，又不会产生任何污染及感染风险。
本发明的目的是提供一种拭子样本核酸释放剂。
本发明另一目的是提供所述拭子样本核酸释放剂的应用。
本发明上述目的通过以下技术方案实现：
一种拭子样本核酸释放剂，包括如下组分：
(1)1-20mol/L金属离子螯合剂；
(2)pH 6-9、5-80mmol/L缓冲液；
(3)5％-40％(v/v)极性有机溶剂；
(4)0.2％-10％(v/v)表面活性剂；
(5)0.1-1mol/L核酸酶抑制剂。
其中，优选地，所述金属离子螯合剂包括但不限于EDTA、EGTA或柠檬酸中的一种或几种。
优选地，所述缓冲液包括但不限于Tris缓冲液、柠檬酸钠缓冲液、磷酸钠缓冲液或醋酸钠缓冲液。
优选地，所述极性有机溶剂包括但不限于DMSO、六甲基磷酰胺、四甲基乙二胺中的一种或几种。
优选地，所述表面活性剂包括但不限于Triton X-100、NP-40、Tween-20或SDS中的一种或几种。
优选地，所述核酸酶抑制剂包括但不限于盐酸胍、异硫氰酸胍或4,4’-羰基双(2-(1-萘酰胺)苯甲酸)。
优选地，所述金属离子螯合剂的浓度为5mol/L，所述缓冲液的浓度为50mmol/L、pH8.5。
优选地，所述极性有机溶剂的浓度为25％。
优选地，所述表面活性剂的浓度为2％。
优选地，核酸酶抑制剂的浓度为0.3mol/L。
本发明所述样本释放剂的使用方法为：将拭子放入装有所述样本释放剂的容器中，室温条件下，将所述容器上下颠倒5-10次或混匀20-40秒，静置5-20分钟(优选5-10分钟)，再次上下颠倒5-10次或混匀20-40秒，瞬时离心后，上清即为释放后的核酸溶液，所得核酸溶液即可用于PCR扩增。拭子样本在样本释放剂中保存、运输、并完成核实释放过程，全程无需开盖，安全、高效，使用操作极简，应用十分广泛。
因此，所述样本释放剂在制备拭子样本核酸释放试剂盒中的应用，以及所开发的含有所述样本释放剂的拭子样本核酸释放试剂盒，均应在本发明的保护范围之内。
本发明具有以下有益效果：
本发明开发的样本释放剂可以集取样保存-灭活-核酸提取三步为一步。取样后可直接放入装有样本释放剂的采样管中进行保存、运输；在使用时将采样管上下颠倒混匀，样本与样本释放剂充分混匀后，常温静置15分钟，即可在灭活样本的同时，将样本的核酸释放出来。整个操作过程不用加热及中途开盖，有效的避免了对于实验操作人员及实验室的污染，最大程度上简化了核酸提取的操作。
本发明的样本释放剂至少存在以下几个方面的优势：
(1)具有样本保存功能：样本核酸释放完成后，能够持久的保护核酸不降解，常温条件下可保存核酸72小时不降解；
(2)不需预处理，直接进行样本的常温灭活、裂解，实现核酸释放：
(3)可直接用于PCR扩增，无需再预处理，不影响后续PCR效果：
(4)使用操作极其简便，不依赖任何外部仪器，无污染，核酸损失极低：采集样本后，只需要上下颠倒5-10次或振荡30秒，对于临床样本的灭活，裂解及核酸的释放均在同一管内进行；
(5)耗时短：样本采集完成后，只需15分钟即可完成对样本的核酸释放，瞬时离心后即可得到核酸溶液；
(6)对实验室和实验操作人员的污染风险小：整个核酸释放的过程均在室温下进行，可有效降低气溶胶的污染，能有效的保护实验操作人员。
因此相比于传统核酸检测，本发明是一个很好的补充和替代方案，在突发性传染病事件中，本发明的样本释放剂对于样本的采集-保存-运输-灭活-核酸提取，有着快速、安全、稳定、高效、成本低等明显的优势。
注：本文中提及的“灭活”是指：释放剂所含的极性有机溶剂、表面活性剂，理论上能在一定程度上破裂细胞、变性蛋白，使病毒失去活性而不具备传染能力。
附图说明
图1为本发明样本释放剂和市购某公司释放剂提取新冠临床样本1、2上机荧光定量PCR扩增图。
图2为本发明样本释放剂和市购某公司释放剂提取新冠临床样本1、2上机荧光定量PCR扩增图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1
一种拭子样本核酸释放剂，包括如下组分：
(1)金属离子螯合剂：5mol/L EDTA；
(2)缓冲液：pH8.5、50mmol/L Tris缓冲液；
(3)极性有机溶剂：25％DMSO；
(4)表面活性剂：2％Triton X-100；
(5)核酸酶抑制剂：0.3mol/L盐酸胍。
2、上述样本释放剂的使用方法为：将采集好的鼻咽拭子放入装有样本释放剂的样本采集管中，室温条件下，将含有样本的采样管手动上下颠倒混匀30秒，静置5分钟，再次上下颠倒30秒，瞬时离心后，样本核酸即释放到样本释放剂中，上清即为释放后的核酸溶液。
所得核酸溶液可直接吸取加入PCR反应液中，上PCR扩增仪进行扩增。
实施例2
一种拭子样本核酸释放剂，包括如下组分：
(1)金属离子螯合剂：10mol/L EGTA；
(2)缓冲液：pH8、60mmol/L柠檬酸钠缓冲液；
(3)极性有机溶剂：30％DMSO；
(4)表面活性剂：5％NP-40；
(5)核酸酶抑制剂：0.5mol/L异硫氰酸胍。
实施例3
一种拭子样本核酸释放剂，包括如下组分：
(1)金属离子螯合剂：1mol/L柠檬酸；
(2)缓冲液：pH 9、40mmol/L磷酸钠缓冲液；
(3)极性有机溶剂：40％六甲基磷酰胺；
(4)表面活性剂：1％SDS；
(5)核酸酶抑制剂：1mol/L 4,4’-羰基双(2-(1-萘酰胺)苯甲酸)。
实施例4
一种拭子样本核酸释放剂，包括如下组分：
(1)金属离子螯合剂：20mol/L EDTA；
(2)缓冲液：pH 6、80mmol/L醋酸钠缓冲液；
(3)极性有机溶剂：5％四甲基乙二胺；
(4)表面活性剂：10％Tween-20；
(5)核酸酶抑制剂：1mol/L盐酸胍。
实施例5
一种拭子样本核酸释放剂，包括如下组分：
(1)金属离子螯合剂：1mol/L EDTA；
(2)缓冲液：pH 9、5mmol/L Tris缓冲液；
(3)极性有机溶剂：40％DMSO；
(4)表面活性剂：2％SDS、2％Tween-20；
(5)核酸酶抑制剂：0.1mol/L盐酸胍。
实施例6
1、实验材料
(1)实施例1-5的拭子样本核酸释放剂；
(2)市购某公司释放剂：其主要成分包括：0.1mmol/L的十二烷基硫酸锂(表面活性剂)，50mmol/L的KCl，0.1％(W/V)的SDS，1％(V/V)乙醇。
2、以实施例1-5的拭子样本核酸释放剂，分别对2020年1～3月广州地区收集的新型冠状病毒临床阳性样本1、2(由云康临床检验中心提供)进行提取。所得核酸溶液作为模板，直接吸取加入PCR反应液中，上PCR扩增仪进行扩增。
同时以市购某公司释放剂为对照。取同时平行取样的拭子临床阳性样本按照厂家说明书进行核酸提取，取提取后的核酸PCR检测。
其中核酸PCR检测所使用的PCR扩增试剂盒是中山大学达安基因股份有限公司新型冠状病毒2019-nCoV核酸检测试剂盒(荧光PCR法)(国械准注20203400063)。
PCR体系如表1：
表1
扩增体系 模板量 总体积
20 5 25
具体PCR扩增程序如表2：
表2
Figure PCTCN2020124134-appb-000001
2、扩增结果如表3及图1-2所示，以实施例1的扩增结果为例展示。
结果显示，本发明的样本释放剂提取新冠临床样本Ct值比市场某公司样本释放剂提取相同样本靠前约1-3个Ct，本发明样本释放剂提取效果显著优于对照释放剂。
表3
Figure PCTCN2020124134-appb-000002
Figure PCTCN2020124134-appb-000003
实施例7
释放剂稳定性测试(对样本的保存效果)：
临床样本：2020年1～3月广州地区收集的新型冠状病毒临床阳性样本3、4(由云康临床检验中心提供)
以实施例1为例，利用本发明样本释放剂对新型冠状病毒临床样本3、4进行保存，分别在室温放置3天、4℃放置7天、-20℃放置15天，然后进行荧光定量PCR扩增，PCR检测所使用的PCR扩增试剂盒是中山大学达安基因股份有限公司新型冠状病毒2019-nCoV核酸检测试剂盒(荧光PCR法)(国械准注20203400063)。
Ct值数据如表4-表6所示，核酸稳定保存，表明无论是低温-20℃、4℃，还是常温，本发明样本释放剂对核酸样本的保存效果也非常好。
表4 放置一段时间(室温3天)后：
Figure PCTCN2020124134-appb-000004
Figure PCTCN2020124134-appb-000005
表5 放置一段时间(4℃7天)后：
Figure PCTCN2020124134-appb-000006
表6 放置一段时间(-20℃15天)后：
Figure PCTCN2020124134-appb-000007
实施例8
1、临床样本
2020年1～3月广州地区收集的新型冠状病毒临床阳性样本3(由云康临床检验中心提供)
2、实验材料
(1)实验组：实施例1的样本释放剂
(2)对比组1，样本释放剂组成如下：
缓冲液：pH8.5、50mmol/L Tris缓冲液；
极性有机溶剂：25％DMSO；
表面活性剂：2％Triton X-100；
核酸酶抑制剂：0.3mol/L盐酸胍。
(3)对比组2，样本释放剂组成如下：
金属离子螯合剂：5mol/L EDTA；
缓冲液：pH8.5、50mmol/L Tris缓冲液；
核酸酶抑制剂：0.3mol/L盐酸胍。
(4)对比组3，样本释放剂组成如下：
金属离子螯合剂：5mol/L EDTA；
缓冲液：pH8.5、50mmol/L Tris缓冲液；
表面活性剂：2％Triton X-100；
核酸酶抑制剂：0.3mol/L盐酸胍。
(5)对比组4，样本释放剂组成如下：
缓冲液：pH8.5、50mmol/L Tris缓冲液；
表面活性剂：2％Triton X-100；
核酸酶抑制剂：0.3mol/L盐酸胍。
(6)对比组5，样本释放剂组成如下：
金属离子螯合剂：5mol/L EDTA；
极性有机溶剂：25％DMSO；
表面活性剂：2％Triton X-100；
核酸酶抑制剂：0.3mol/L盐酸胍。
(7)对比组6，样本释放剂组成如下：
金属离子螯合剂：5mol/L EDTA；
缓冲液：pH8.5、50mmol/L Tris缓冲液；
极性有机溶剂：25％DMSO；
核酸酶抑制剂：0.3mol/L盐酸胍。
3、实验方法
以实施例1为例，利用本发明样本释放剂处理新型冠状病毒临床样本3，然后进行荧光定量PCR扩增，PCR检测所使用的PCR扩增试剂盒是中山大学达安基因股份有限公司新型冠状病毒2019-nCoV核酸检测试剂盒(荧光PCR法)(国械准注20203400063)。
4、扩增结果如表7。结果显示，本发明样本释放剂提取的新冠临床样本Ct值比对比组提取相同样本靠前约3-6个Ct，本发明样本释放剂提取效果显著优于对照释放剂。
表7 荧光定量PCR扩增Ct值(靶标N基因)
实验组 26.0
对比组1 29.0
对比组2 30.3
对比组3 30.8
对比组4 30.5
对比组5 28.7
对比组6 27.9
5、另外将上述利用实验组和对比组1-6释放剂提取的核酸样本在室温放置3天后进行荧光定量PCR扩增，Ct值数据如表8所示。
表8 核酸样本室温放置3天后对靶标N基因的检测Ct值
Figure PCTCN2020124134-appb-000008
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施 例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (11)

  1. 一种拭子样本核酸释放剂，其特征在于，包括如下组分：
    (1)1-20mol/L金属离子螯合剂；
    (2)pH 6-9、5-80mmol/L缓冲液；
    (3)5％-40％(v/v)极性有机溶剂；
    (4)0.2％-10％(v/v)表面活性剂；
    (5)0.1-1mol/L核酸酶抑制剂。
  2. 根据权利要求1所述样本释放剂，其特征在于，所述金属离子螯合剂为EDTA、EGTA或柠檬酸中的一种或几种。
  3. 根据权利要求1所述样本释放剂，其特征在于，所述缓冲液为Tris缓冲液、柠檬酸钠缓冲液、磷酸钠缓冲液或醋酸钠缓冲液。
  4. 根据权利要求1所述样本释放剂，其特征在于，所述极性有机溶剂为DMSO、六甲基磷酰胺、四甲基乙二胺中的一种或几种。
  5. 根据权利要求1所述样本释放剂，其特征在于，所述表面活性剂为Triton X-100、NP-40、Tween-20或SDS中的一种或几种；所述核酸酶抑制剂为盐酸胍、异硫氰酸胍或4,4’-羰基双(2-(1-萘酰胺)苯甲酸)。
  6. 根据权利要求1所述样本释放剂，其特征在于，所述金属离子螯合剂的浓度为5mol/L，所述缓冲液的浓度为50mmol/L、pH8.5。
  7. 根据权利要求1所述样本释放剂，其特征在于，所述极性有机溶剂的浓度为25％，所述表面活性剂的浓度为2％，核酸酶抑制剂的浓度为0.3mol/L。
  8. 一种拭子样本保存与核酸释放方法，其特征在于，将拭子放入装有权利要求1-7任一所述样本释放剂的容器中，室温条件下保存；需要提取核酸时，将所述容器上下颠倒混匀5-10次或混匀20-40秒，静置5-20分钟，再次上下颠倒5-10次或混匀20-40秒，瞬时离心后，上清即为释放后的核酸溶液。
  9. 根据权利要求8所述方法，其特征在于，室温条件下保存的时间为0-72h。
  10. 权利要求1-7任一所述样本释放剂在制备拭子样本核酸释放试剂盒中的的应用。
  11. 一种拭子样本核酸释放试剂盒，其特征在于，含有权利要求1-7任一所述样本释放剂。
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