WO2023179216A1 - 一种生物样本提取液及其应用和使用方法 - Google Patents

一种生物样本提取液及其应用和使用方法 Download PDF

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WO2023179216A1
WO2023179216A1 PCT/CN2023/074724 CN2023074724W WO2023179216A1 WO 2023179216 A1 WO2023179216 A1 WO 2023179216A1 CN 2023074724 W CN2023074724 W CN 2023074724W WO 2023179216 A1 WO2023179216 A1 WO 2023179216A1
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biological sample
virus
extraction
surfactant
samples
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French (fr)
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商涛
费凤琴
孔健希
郑珍珍
童芳丽
吴思璐
崔亚茹
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艾康生物技术(杭州)有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biological detection technology, and particularly relates to virus sample preservation solutions, usage methods and applications.
  • Viruses are non-cellular organisms that are small in size and have a simple structure. They must be parasitic in living cells and multiply by replication. It consists of a protein shell and the genetic material nucleic acid (DNA or RNA) inside. Viruses cannot survive independently and must live within the cells of other organisms.
  • the virus in the sample is decomposed using the extraction solution, and then the nucleic acid detection method is used to analyze the decomposed nucleic acid material, and the antigen detection method is used to analyze the protein obtained from the decomposition to determine whether the corresponding virus is present in the sample.
  • SARS-CoV-2 coronavirus
  • inactivating agents such as guanidinium salts
  • guanidine salts can cause protein denaturation and are not suitable for antigen detection methods that use proteins as detection objects.
  • the new coronavirus antigen test has been promoted and used among community populations because of its simple detection operation and rapid results. It can achieve the purpose of rapid population screening through self-testing of community populations. How these non-professionals collect and test the remaining samples can be quickly and effectively inactivated to avoid exposure to these infectious and pathogenic virus samples. This is what developers of virus antigen detection reagents need to study in depth and develop a method that can test virus samples. Effectively inactivated extracts are of great significance.
  • the present invention provides an extract of a biological sample (virus), including a surfactant and salt ions, and the surfactant is selected from Triton X-100 or Tween-20.
  • the concentration of the surfactant is 0.5% to 2% (w/V).
  • the salt ion concentration is 0.9%-5% (w/V).
  • the salt ion is selected from NaCl or KCl.
  • the pH value of the extraction solution is 8.0-9.0.
  • the invention also provides the application of the virus extract in extracting viral protein samples.
  • the biological sample is a virus
  • the virus is selected from novel coronavirus.
  • the invention also provides a method for using the biological sample extract, which includes the following steps: directly adding swab samples or liquid samples to a certain volume of virus extract, incubating and extracting, and adding the extracted sample dropwise to the reagent. On the board, the results are interpreted.
  • the swab sample or liquid sample is incubated in the virus extraction solution for 30 seconds to 1 minute.
  • the reagent plate includes an upper cover and a lower plate, and a test strip is assembled between the upper cover and the lower plate.
  • the biological sample extract of the present invention can not only improve the detection sensitivity of immunochromatographic antigen detection products, but also improve the inactivation of viruses (such as novel coronavirus (SARS-CoV-2) and other viruses) by the extract. ability. It is conducive to quickly and effectively inactivating samples collected by non-professionals, avoiding the exposure of infectious and pathogenic virus samples, and plays an important role in preventing secondary contamination and protecting the population.
  • viruses such as novel coronavirus (SARS-CoV-2) and other viruses
  • Figure 1 is a microscopic examination photo of the negative control in Example 2.
  • Figure 2 is a microscopic examination photo of the positive control in Example 2.
  • Figure 3 is a microscopic examination photo of the experimental group of Example 2.
  • Figure 4 contains the extraction solution in the extraction tube.
  • Figure 5 Reagent plate installed in extraction tube.
  • the invention provides an extraction liquid with an inactivation effect, which includes surfactant and salt ions.
  • the surfactant is selected from, but not limited to, Triton X-100 or Tween-20
  • the salt ion is selected from, but is not limited to, sodium chloride or potassium chloride.
  • the salt ions in the extraction solution are of high concentration salt ions.
  • the surfactant is selected from Triton X-100 or Tween-20 or SDS.
  • the salt ion is selected from NaCl or KCl.
  • New coronavirus antigen detection kit new coronavirus (2019-nCoV) antigen detection kit (latex method) provided by Aikang Company)
  • step 3 Use the highly sensitive formula in step 2 to prepare an extraction solution that dilutes the new coronavirus strain to a concentration of 1.0*10 6 TCID 50 /mL.
  • the reagent plate includes an upper cover and a lower plate, and a test strip is assembled between the upper cover and the lower plate.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Virology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种生物样本提取液及其应用和使用方法，生物样本提取液包括表面活性剂和盐离子，表面活性剂选自Triton X-100或Tween-20。生物样本提取液不仅可以提高免疫层析抗原检测产品的检测灵敏度，同时还提高了提取液对新型冠状病毒等各种病毒的灭活能力，为防止二次污染和对人群的保护起到了重要的作用。

Description

一种生物样本提取液及其应用和使用方法 技术领域
本发明属于生物检测技术领域，尤其涉及病毒样本保存液、使用方法和应用。
背景技术
病毒(Biological virus)是一种个体微小、结构简单，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。它由蛋白质外壳和内部的遗传物质核酸(DNA或RNA)组成。病毒不能独立生存，必须生活在其他生物的细胞内。
当前病毒检测通常采用核酸检测法或抗原抗体检测法。利用提取液将样本中的病毒分解，然后采用核酸检测法对分解出来的核酸物质进行分析，采用抗原检测法对分解获得的蛋白质进行分析，以判断样本中是否存在相应病毒。
以目前流行的新型冠状病毒(SARS-CoV-2)检测为例。利用拭子在口、鼻等特定部位采集样本，然后将采集了样本的拭子浸没在病毒提取液中，以备后续检测分析用。
完成检测后，保存有病毒样本的提取液通常还有剩余。在处理这些剩余提取液的过程中，采集的样本是否完全灭活是检测过程中面临的一个风险。未能完全灭活的病毒样品在转运和处理过程中，对工作人员存在二次感染的风险。特别是在针对高传染性、高致病性的新型冠状病毒等病毒的处理上，需要能对样本中的病毒进行有效灭活以阻断因为二次感染导致的疫情传播途径。
直接在提取液中加入胍盐等灭活剂是目前病毒样本灭活的方法之一。但是胍盐会导致蛋白变性，在以蛋白为检测对象的抗原检测方法中并不适用。
而目前新型冠状病毒抗原检测因其检测操作简单，出结果快速的特性，已在社区人群中推广和使用，通过社区人群的自测实现人群快速筛查的目的。这些非专业人员采集和测试剩余的样本如何快速有效地被灭活，避免这些传染性和致病性的病毒样本暴露，是病毒抗原检测试剂开发者需要深入研究的，开发出能对病毒样本进行有效灭活的提取液具有重要意义。
发明内容
为了解决现有技术的上述问题，本发明提供了一种生物样本(病毒)的提取液，包括表面活性剂和盐离子，所述表面活性剂选自Triton X-100或Tween-20。
进一步的，所述表面活性剂的浓度为0.5％～2％(w/V)。
进一步的，所述盐离子浓度为0.9％-5％(w/V)。
进一步的，所述盐离子选自NaCl或KCl。
进一步的，提取液的pH值为8.0-9.0。
本发明还提供了所述病毒提取液在病毒蛋白样本提取中的应用。
进一步的，所述生物样本为病毒，更进一步的，所述病毒选自新型冠状病毒。
本发明还提供了所述生物样本提取液的使用方法，包括如下步骤：直接将拭子样本或液体样本加入到一定体积的病毒提取液中，孵育提取，将提取处理后的样本滴加在试剂板上，结果判读。
进一步的，所述拭子样本或液体样本在病毒提取液中孵育30秒至1分钟。
进一步的，所述试剂板包括上盖和下板，在上盖和下板之间装配试纸条。
本发明所述生物样本提取液不仅可以提高免疫层析抗原检测产品的检测灵敏度，同时还提高了提取液对病毒(例如：新型冠状病毒(SARS-CoV-2)等各种病毒)的灭活能力。有利于快速有效地灭活非专业人员自行采集的样本，避免具传染性和致病性的病毒样本暴露，为防止二次污染和对人群的保护起到了重要的作用。
附图说明
图1实施例2阴性对照的显微镜镜检照片。
图2实施例2阳性对照的显微镜镜检照片。
图3实施例2实验组的显微镜镜检照片。
图4装在提取管中提取液。
图5装在提取管中试剂板。
图6拭子采样和检测步骤。
具体实施方式
以下结合具体实施例，对本发明做进一步说明。但以下实施例仅用于说明本发明，并不是用于限制本发明的范围。
本发明提供了一种具有灭活效果的提取液，所述提取液包括表面活性剂和盐离子。所述表面活性剂选自但不限于Triton X-100或Tween-20，所述盐离子选自但不限于氯化钠或氯化钾。在一个优选的实例中，提取液中的盐离子为高浓度的 盐离子。
实施例1
实验材料：
1、提取液配方：
表面活性剂选自Triton X-100或Tween-20或SDS。
盐离子选自NaCl或KCl。
2、新冠抗原检测试剂盒(由艾康公司提供的新型冠状病毒(2019-nCoV)抗原检测试剂盒(乳胶法))
3、新冠病毒N蛋白重组抗原
4、新冠病毒株
实验方法：
1.将表面活性剂Triton X-100、Tween-20、SDS和盐离子NaCl、KCl按不同比例与纯化水配制成不同浓度的提取液。提取液的pH值为8.0-9.0。具体配方如表1所示。
表1提取液配方表

*(m/V)表示每100ml溶剂中溶解的溶质的克数
2.分别用表1配方的提取液配置相同浓度(低浓度和中浓度)的新冠病毒N蛋白重组抗原质控品，并用新冠抗原检测试剂盒进行灵敏度检测。其中所述低浓度：新冠病毒N蛋白重组抗原的浓度为20pg/mL，所述中浓度：新冠病毒N蛋白重组抗原的浓度为60pg/mL。实验结果见表2。
表2：灵敏度实验结果

*L表示显色强度，从L1到L10，后面的数值越高，表示产品测试线的显色
越强。低浓度的L值要达到L3，中浓度的L值越高越好。
3.选用步骤2中灵敏度高的配方配制提取液，所述提取液将新冠病毒株稀释至浓度1.0*106TCID50/mL。
4.在室温下孵育60分钟后，将提取液处理后的病毒按照1：10000的比例用培养基进行稀释，作为实验组。不加病毒的提取液按1：10000的比例用培养基进行稀释，作为阴性对照组，将浓度为1.0*106TCID50/mL的病毒直接按1：10000比例用培养基进行稀释，作为阳性对照组。
5.将培养板培养孔内的Vero E6细胞株培养至80％密度左右，将步骤4的实验组、阴性对照组和阳性对照组的病毒溶液转移到所述培养孔中，将培养板放置在37℃，5％CO2培养箱中培养3天。
6.观察细胞的生产状态，评估提取液的灭活影响。
实施例2提取液的灭活实验
选用实施例1的配方7作为提取液，按实施例1的方法进行病毒的灭活实验，实验结果如下表3。
表3病毒灭活实验结果

结果解释：N/D为未发现病毒；CPE为病毒存活；Non-CPE为病毒灭活。
选用灵敏度高的其他配方提取液进行灭活实验，具有同样的灭活效果。
实施例3
实验步骤如下：如图4、图5和图6所示，打开装有提取液1的提取管2，所述提取管中的提取液采用表1的配方7,；将采样后的拭子3插入到含有提取液的提取管中，在提取管内旋转拭子30秒，然后将拭子头部在管壁上挤压5次，确保拭子上的样本充分洗脱；沿提取管内壁挤压拭子头部，使提取液尽可能保留在管内，取出并弃去拭子，得处理后的样本提取物；将滴头4盖入提取管；向试剂板5中滴入4-5滴处理后样本提取物于试剂板加样口7；检测结果判读，检测结果显示在试剂板的观察窗6中。所述试剂板包括上盖和下板，在上盖和下板之间装配有试纸条。
用拭子采集经核酸检测确认的阳性患者和阴性人员的样本，并按本实施例4所述式样步骤进行采样、提取液提取和试剂板加样检测。测试结果和核酸检测结果一致。
本发明所述的提取液中加入0.5％～2％(w/V)的Triton X-100或Tween 20等表面活性剂以及0.9％-5％(w/V)的高浓度的盐离子(如NaCl，KCl)后，不仅可以提高免疫层析产品的检测灵敏度，同时还可以在一定时间内(例如60分钟内)实现对病毒的灭活。为防止剩余样本的二次污染，并对人群的保护起到了重要的作用。

Claims (10)

  1. 一种生物样本提取液，其特征在于，包括表面活性剂和盐离子，所述表面活性剂选自Triton X-100或Tween-20。
  2. 根据权利要求1所述的生物样本提取液，其特征在于，所述表面活性剂的浓度为0.5％～2％(w/V)。
  3. 根据权利要求2所述的生物样本提取液，其特征在于，所述盐离子浓度为0.9％-5％(w/V)。
  4. 根据权利要求1所述的生物样本提取液，其特征在于，所述盐离子选自NaCl或KCl。
  5. 根据权利要求1所述的生物样本提取液，其特征在于，提取液的pH值为8.0-9.0。
  6. 根据权利要求1至5之一所述生物样本提取液在病毒蛋白样本提取中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述病毒选自新型冠状病毒。
  8. 根据权利要求1至5之一所述生物样本提取液的使用方法，其特征在于，直接将拭子样本或液体样本加入到一定体积的病毒提取液中，孵育提取，将提取处理后的样本滴加在试纸上，判读结果。
  9. 根据权利要求8所述的使用方法，其特征在于，所述拭子样本或液体样本在病毒提取液中孵育30秒至1分钟。
  10. 根据权利要求8所述的使用方法，其特征在于，所述试剂板包括上盖和下板，在上盖和下板之间装配有试纸条。
PCT/CN2023/074724 2022-03-25 2023-02-07 一种生物样本提取液及其应用和使用方法 WO2023179216A1 (zh)

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