WO2022110335A1 - 一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法 - Google Patents

一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法 Download PDF

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WO2022110335A1
WO2022110335A1 PCT/CN2020/136280 CN2020136280W WO2022110335A1 WO 2022110335 A1 WO2022110335 A1 WO 2022110335A1 CN 2020136280 W CN2020136280 W CN 2020136280W WO 2022110335 A1 WO2022110335 A1 WO 2022110335A1
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朱海红
陈超
任艳丽
蔡萧鹏
陈智
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Zhejiang University
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  • the invention belongs to the field of clinical medical diagnosis, and relates to the diagnosis of viral hepatitis B, and the fields of infectious diseases, molecular biology, cell biology and the like. More specifically, the present invention relates to a nucleic acid amplification detection method of hepatitis B virus (Hepatitis B virus, HBV) gene in blood, and provides a kit for rapid detection of hepatitis B virus gene and a detection method thereof.
  • Hepatitis B virus Hepatitis B virus, HBV
  • Hepatitis B is an infectious disease mainly caused by liver lesions caused by hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • liver function damage, loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue are the main manifestations.
  • Some patients may have jaundice and fever.
  • Some hepatitis B patients can become chronic, and even develop into liver cirrhosis, and a few can develop into liver cancer. Mainly through blood and blood products, mother-to-child transmission and sexual contact.
  • HBV belongs to the genus Ortho Hepadnavirus of the family Hepadnaviridae. Three kinds of particles with different shapes were observed in the serum of HBV infected patients under electron microscope. The first is a small spherical hollow particle with a diameter of 22nm, and the second is a tubular particle of 22nmx40-100nm. Both particles are enveloped components and do not contain viral nucleic acid. The third is spherical particles with a diameter of 42 nm, called Dane particles, which are composed of an envelope, a core-shell and a core.
  • the envelope is composed of HBsAg; the nucleocapsid is composed of hepatitis B core antigen (HBcAg); the core contains part of circular double-stranded DNA and DNA polymerase.
  • the HBV genome is a partially circular double-stranded DNA consisting of positive and negative strands, and the negative strand is a long chain. It is a closed circular DNA with a total length of about 3200 nucleotides.
  • the positive strand is a short, semi-closed circular DNA, and the full length is 50%-75% of the negative strand.
  • Fluorescence quantitative PCR is a nucleic acid quantitative detection technology that integrates PCR technology, fluorescence signal detection and data analysis. Fluorescence quantitative PCR uses specific probes, which can specifically identify target sequences, have dual control of primers and probes, and integrate fluorescent labeling technology, laser detection technology, and digital imaging technology, with high specificity and sensitivity. characteristics, high accuracy and low false positive rate.
  • the thermal cycling module and the fluorescence detection module are indispensable components of real-time quantitative PCR, so real-time quantitative PCR often requires expensive and bulky special instruments. This makes real-time PCR unsuitable for use in non-laboratory settings.
  • Recombinase polymerase amplification (RPA) technology is an isothermal amplification technology that can be applied to low temperature (about 37°C).
  • Enzymes, DNA polymerases with strand displacement activity, single strand DNA-binding proteins (SSB) and Mg2+, etc. use primer-recombinase complexes to scan DNA strands, thereby promoting the corresponding homologous sites on the DNA strands.
  • the identification, binding, and exchange of dots synthesize complementary new strands through the combined action of DNA polymerase and SSB. As the reaction proceeds, the amplification product grows exponentially.
  • RPA can be used as a portable, fast and cost-effective detection tool to achieve accurate on-site detection outside the laboratory.
  • the invention provides a detection kit for hepatitis B virus with stronger sensitivity and specificity and a detection method thereof.
  • the present invention provides primers for rapid detection of hepatitis B virus, the primers are recombinase polymerase amplification DNA primers:
  • the primer is used for the preparation of a kit for rapid detection of hepatitis B virus.
  • a kit for rapid detection of hepatitis B virus including: recombinase polymerase amplification system and Cas13 reaction system; the recombinase polymerase amplification system includes TwistAmp liquid recombinase polymerase amplification kit and recombinase polymerase Enzymatic amplification of DNA primers:
  • the Cas13 reaction system includes recombinant CRISPR-Cas13a protein, single-stranded guide RNA and single-stranded RNA probe.
  • the recombinase polymerase amplification system includes:
  • the Cas13 reaction system includes:
  • the method for detecting hepatitis B virus gene by the kit includes the following steps:
  • Detection use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
  • the RPA of the present application is combined with the gene editing system of CRISPR/Cas13a, which can detect hepatitis B virus in a portable, rapid and precise manner with good sensitivity and specificity.
  • Fig. 1 Example 1 change curve of fluorescence intensity with time.
  • Fig. 2 The effect of adding template concentration on fluorescence intensity in Example 1.
  • TwistAmp liquid recombinase polymerase amplification kit including 20x core reaction mixture, 2x reaction buffer, 280mM magnesium acetate (MgOAc), 10x electron mixture, purchased from TwistDx Company.
  • Trizma hydrochloric acid buffer-400mM Tris pH 7.4 (Sigma Aldrich).
  • NxGen T7 RNA polymerase purchased from Lucigen Company.
  • Detection use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.

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Abstract

一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法，涉及乙型病毒性肝炎的诊断，以及感染病学、分子生物学和细胞生物学领域。该试剂盒及检测方法将RPA与CRISPR/Cas13a的基因编辑系统相结合，可以便携、快速、精确地检测乙型肝炎病毒，具有很好的灵敏度和特异性。

Description

一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法 技术领域
本发明属临床医学诊断领域，涉及乙型病毒性肝炎的诊断，以及感染病学、分子生物学和细胞生物学等领域。更具体地，本发明涉及血液中乙型肝炎病毒(Hepatitis B virus，HBV)基因的核酸扩增检测方法，提供了一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法。
背景技术
乙型肝炎是由乙型肝炎病毒(Hepatitis B virus，HBV)感染引起的以肝脏病变为主的一种传染病。临床上以肝功能损害、食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现，部分患者可有黄疸发热。部分乙肝患者可慢性化，甚至发展成肝硬化，少数可发展为肝癌。主要通过血和血制品、母婴传播及性接触传播。
HBV属嗜肝DNA病毒科(Hepadnaviridae)正嗜肝DNA病毒属(Ortho Hepadnavirus)。HBV感染者血清中电镜下可见3种不同形态的颗粒。第一种为直径22nm的小球形空心颗粒，第二种为22nmx40-100nm的管状颗粒。这两种颗粒均为包膜成分，不含病毒的核酸。第三种为直径42nm的球形颗粒，称Dane颗粒，由包膜、核壳和核心组成。包膜由HBsAg组成；核壳由乙型肝炎核心抗原(HBcAg)组成；核心内有部分环状双链DNA和DNA聚合酶。
HBV基因组为部分环状双链DNA，由正链和负链组成，负链为长链。呈闭合型的环状DNA，全长约3200个核苷酸。正链为短链，呈半闭合型的环状DNA，全长为负链的50％-75％。
目前，常用HBV-DNA检测方法主要是荧光定量PCR法。荧光定量PCR法是一种集PCR技术、荧光信号检测和数据分析于一体的核酸定量检测技术。荧光定量PCR使用了特异性探针，能够对靶序列进行特异性的识别，具有引物探针双重控制，且综合了荧光标记技术、激光检测技术、数码显像技术，具有高特异性、高灵敏性，高准确性及低假阳性率等特点。其中热循环模块和荧光检测模块是荧光定量PCR不可缺少的组成部分，因此荧光定量PCR往往需要昂贵笨重的特殊仪器。这使得荧光定量PCR不适合在非实验室环境中使用。
重组酶聚合酶扩增技术(Recombinase polymerase amplification，RPA)技术是一种可应用于低温(37℃左右)的等温扩增技术，反应体系包括能结合单链核酸(寡核苷酸引物)的重组酶、具有链置换活性的DNA聚合酶、DNA单链结合蛋白(single strand DNA-binding protein，SSB)和Mg2+等，利用引物-重组酶复合物扫描DNA链，进而促进DNA链上对应同源位点的识别、结合、互换，通过DNA聚合酶和SSB共同作用合成互补新链，随着反应的进行，扩增产物以指数状态增长。RPA可作为一种便携、快速、性价比高的检测工具，实现实验室外的现场精确检测。
发明内容
本发明提供了一种灵敏度和特异性更强的检测乙型肝炎病毒的试剂盒及其检测方法。
本发明提供用于快速检测乙型肝炎病毒的引物，所述引物为重组酶聚合酶扩增DNA引物：
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′。
所述引物用于制备快速检测乙型肝炎病毒的试剂盒的应用。
用于快速检测乙型肝炎病毒的试剂盒，包括：重组酶聚合酶扩增体系和Cas13反应体系；所述重组酶聚合酶扩增体系包括TwistAmp液态重组酶聚合酶扩增试剂盒和重组酶聚合酶扩增DNA引物：
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTT-CTG-3′
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′
所述Cas13反应体系包括重组CRISPR-Cas13a蛋白、单链引导RNA和单链RNA探针。
单链引导RNA：
5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′
单链RNA探针：
5′-FAM-TUUUUUC-BHQ-1-3′。
具体的，
所述重组酶聚合酶扩增体系包括：
Figure PCTCN2020136280-appb-000001
所述Cas13反应体系包括：
Figure PCTCN2020136280-appb-000002
试剂盒检测乙型肝炎病毒基因的方法，包括如下步骤：
1)重组酶聚合酶扩增
(1)取HBV阳性对照稀释成3x10 5拷贝/μl，3x10 4拷贝/μl，3x10 3拷贝/μl，3x10 2拷贝/μl，3x10拷贝/μl，取1μl加入以下反应体系：
Figure PCTCN2020136280-appb-000003
(2)加入1.25μl的20x核心反应混合液，混匀离心；
(3)加入1μl模板(不同稀释度的质粒模板和阴性对照)；
(4)在管盖上加入1.25μl乙酸镁(MgOAc，280mM)；
(5)颠倒混匀，离心后39度孵育20min。
2)用Cas13蛋白检测HBV
(1)取上述1ul反应产物加入Cas13反应体系，加入384孔透明圆底酶标板充分混合。设3个复孔：
Figure PCTCN2020136280-appb-000004
Figure PCTCN2020136280-appb-000005
(2)反应时间30min。
(3)检测：用多功能酶标仪检测仪，检测480nm激发光520nm发射波长的荧光强度。
本申请RPA与CRISPR/Cas13a的基因编辑系统相结合，可以便携、快速、精确地检测乙型肝炎病毒，具有很好的灵敏度和特异性。
附图说明
图1实施例1随时间荧光强度变化曲线。
图2实施例1加入模板浓度对荧光强度的影响。
具体实施方式
(一)材料
(1)重组酶聚合酶扩增DNA引物：
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′
上述引物均于上海生工生物工程技术服务有限公司合成。
(2)单链引导RNA：
5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′,
于上海生工生物工程技术服务有限公司合成。
(3)单链RNA探针：5′-FAM-TUUUUUC-BHQ-1-3′，
于上海生工生物工程技术服务有限公司合成。
(4)TwistAmp液态重组酶聚合酶扩增试剂盒，包含20x核心反应混合液、2倍反应缓冲液、280mM乙酸镁(MgOAc)、10x电子混合液，购于TwistDx公司。
(5)Trizma盐酸缓冲液-400mM Tris pH 7.4(Sigma Aldrich)。
(6)重组CRISPR-Cas13a蛋白，购于上海惠诚生物科技有限公司。
(7)RNase酶抑制剂,购于上海碧云天生物技术有限公司。
(8)NxGen T7 RNA聚合酶，购于Lucigen公司。
(9)10mM NTP混合液，购于上海生工生物工程技术服务有限公司。
(10)氯化镁溶液(120mM)，用六合氯化镁加ddH 2O配成。
(二)方法：
1.重组酶聚合酶扩增
(1)取HBV阳性对照稀释成3x10 5拷贝/μl，3x10 4拷贝/μl，3x10 3拷贝/μl，3x10 2拷贝/μl，3x10拷贝/μl，取1μl加入以下反应体系：
Figure PCTCN2020136280-appb-000006
(2)加入1.25μl的20x核心反应混合液，混匀离心；
(3)加入1μl模板(不同稀释度的质粒模板和阴性对照)；
(4)在管盖上加入1.25μl乙酸镁(MgOAc，280mM)；
(5)颠倒混匀，离心后39度孵育20min。
2.用Cas13蛋白检测HBV
(1)取上述1ul反应产物加入Cas13反应体系，加入384孔透明圆底酶标板充分混合。设3个复孔：
Figure PCTCN2020136280-appb-000007
Figure PCTCN2020136280-appb-000008
(2)反应时间30min。
(3)检测：用多功能酶标仪检测仪，检测480nm激发光520nm发射波长的荧光强度。
(三)结果
随着加入模板样本浓度的下降，孔内荧光强度逐渐下降，到30分钟时实验组相对于阴性组差异达到较为显著的程度(见图1)。重复进行实验共计3次，统计30分钟时各浓度梯度的荧光强度，可见样本浓度为30拷贝/μl时与对照组仍有明显差异。

Claims (6)

  1. 用于快速检测乙型肝炎病毒的引物，所述引物为重组酶聚合酶扩增DNA引物：
    引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′
    引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′。
  2. 权利要求1所述引物用于制备快速检测乙型肝炎病毒的试剂盒的应用。
  3. 一种用于快速检测乙型肝炎病毒的试剂盒，包括：重组酶聚合酶扩增体系和Cas13反应体系；
    所述重组酶聚合酶扩增体系包括TwistAmp液态重组酶聚合酶扩增试剂盒和重组酶聚合酶扩增DNA引物：
    引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′
    引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′
    所述Cas13反应体系包括重组CRISPR-Cas13a蛋白、单链引导RNA和单链RNA探针单链引导RNA：
    5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′单链RNA探针：
    5′-FAM-TUUUUUC-BHQ-1-3′。
  4. 根据权利要求3所述的试剂盒，其特征在于：所述重组酶聚合酶扩增体系包括：
    Figure PCTCN2020136280-appb-100001
  5. 根据权利要求3所述的试剂盒，其特征在于：所述Cas13反应体系包括：
    Figure PCTCN2020136280-appb-100002
    Figure PCTCN2020136280-appb-100003
  6. 权利要求3-5任一所述试剂盒检测乙型肝炎病毒基因的方法，包括如下步骤：
    1)重组酶聚合酶扩增
    (1)取HBV阳性对照稀释成3x10 5拷贝/μl，3x10 4拷贝/μl，3x10 3拷贝/μl，3x10 2拷贝/μl，3x10拷贝/μl，取1μl加入以下反应体系：
    Figure PCTCN2020136280-appb-100004
    (2)加入1.25μl的20x核心反应混合液，混匀离心；
    (3)加入1μl模板(不同稀释度的质粒模板和阴性对照)；
    (4)在管盖上加入1.25μl乙酸镁(MgOAc，280mM)；
    (5)颠倒混匀，离心后39度孵育20min。
    2)用Cas13蛋白检测HBV
    (1)取上述1ul反应产物加入Cas13反应体系，加入384孔透明圆底酶标板充分混合；设3个复孔：
    Figure PCTCN2020136280-appb-100005
    Figure PCTCN2020136280-appb-100006
    (2)反应时间30min；
    (3)检测：用多功能酶标仪检测仪，检测480nm激发光520nm发射波长的荧光强度。
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