WO2023093387A1 - 用于检测丙型肝炎病毒的raa引物、试剂盒及其应用 - Google Patents

用于检测丙型肝炎病毒的raa引物、试剂盒及其应用 Download PDF

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WO2023093387A1
WO2023093387A1 PCT/CN2022/126394 CN2022126394W WO2023093387A1 WO 2023093387 A1 WO2023093387 A1 WO 2023093387A1 CN 2022126394 W CN2022126394 W CN 2022126394W WO 2023093387 A1 WO2023093387 A1 WO 2023093387A1
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raa
primer
detection
virus
hepatitis
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PCT/CN2022/126394
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French (fr)
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王爱萍
张改平
王海丽
张雨杭
陈玉梅
周景明
张盈
朱习芳
梁超
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郑州大学
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Priority to US18/058,851 priority Critical patent/US20230295752A1/en
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/70—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
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • 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
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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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    • C12Q1/6844—Nucleic acid amplification reactions
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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • 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
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • 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
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB

Definitions

  • the invention relates to the technical field of virus biological detection, in particular to an RAA primer for detecting hepatitis C virus, a kit and an application thereof.
  • Hepatitis C is a blood-borne disease caused by the hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • Departments of science and technology, health and health will include hepatitis C-related research into the scope of the national science and technology plan, strengthen basic and applied research, support research on new prevention and treatment technology strategies, and develop and invest in rapid detection technology to comprehensively promote the construction of a healthy China and protect lay the foundation for people's health.
  • the routine diagnostic methods for hepatitis C are qPCR, PCR and ELISA. Although the three are mature detection methods, they all require expensive equipment, a long detection cycle (3-4 hours), and high cost of reagent consumables. In some municipal CDCs and tertiary medical institutions, the detection capabilities cannot meet the actual needs. There is an urgent need for a nucleic acid detection method that can be operated in ordinary laboratories, does not require high-end equipment, and can achieve rapid detection and direct interpretation.
  • recombinase polymerase amplification recombinase polymerase amplification, RPA; Recombinase Aided Amplification, RAA
  • RPA Recombinase polymerase amplification
  • RAA Recombinase Aided Amplification
  • the RPA/RAA amplification primers are the key to the whole reaction, but there is no software or mature design principles for primer design, and there is no large amount of data to provide a basis for its primer design, let alone mature experience. Therefore, it is imperative to find specific RPA/RAA primers suitable for hepatitis C virus.
  • the object of the present invention is to provide a kind of RAA primer and test kit for detecting hepatitis C virus, to solve the technical problem that prior art is difficult to detect HCV rapidly; And combine it with lateral flow technique, to solve HCV The cost of testing is high, and the results of HCV testing are difficult to present quickly and intuitively.
  • Upstream primer 5'-FITC-CTTGGGATATGATGATGAACTGGTCACCTAC-3' (as shown in SEQ ID NO.1);
  • Downstream primer 5'-Biotin-AAGAGTAGCATCACAATCAGAACCTTAGCC-3' (as shown in SEQ ID NO.2).
  • nucleotide sequences are also included:
  • Upstream primer 5'-FITC-AGTACAGGACTGCAATTGCTCAATATATCC-3'(SEQ ID NO.3);
  • Downstream primer 5'-Biotin-GCAAAGATAGCATCACAATCAGAACCTTAG-3' (SEQ ID NO.4).
  • the RAA primers can be applied to the preparation of hepatitis C virus detection related products, such as:
  • An HCV detection kit is prepared, including RAA primers, RNA extraction reagents, RAA amplification reaction reagents, lateral flow chromatography test strips, positive control substances, negative control substances and the like.
  • RAA amplification system for HCV detection including:
  • the using method of described HCV detection kit comprises the steps:
  • HCV was detected using the recombinase-aided amplification and lateral flow assay (RAA-LFA) gold standard detection card.
  • the RNA of the sample to be tested is extracted with an RNA extraction reagent, and an isothermal amplification reaction is performed using the RAA primer and the corresponding RAA amplification reaction reagent to obtain an RAA amplification product.
  • the isothermal amplification reaction is as follows: add 41.5 ⁇ L of buffer solution, 10 ⁇ L of deionized water, 2.5 ⁇ L of upstream primers and downstream primers, 2 ⁇ L of template positive plasmids, and finally add 280 mmol/L 2.5 ⁇ L of magnesium acetate solution, mix the RAA amplification system thoroughly, place it on a water bath, and react at 30-42°C for 5-30 minutes, and obtain the RAA amplification product after the reaction is completed.
  • a positive control (the positive control adopts the positive plasmid pUC57-pC/E1 as a template) and a negative control (the negative control adopts the empty vector plasmid sample without the genome fragment of hepatitis C virus) are set.
  • the RAA amplification product can be incubated at 37° C. for 5 min, then diluted with 200 ⁇ L of PBST buffer, and added dropwise to the sample well of a colloidal gold-labeled lateral flow test strip, at room temperature Observe the result after incubating for 5-10 minutes. If the test line is red, it is positive (Positive Readout), and if the test line does not develop color, it is negative (Negative Readout).
  • the present invention comprehensively considers many factors such as the target region, the length of the RAA primer and its possibility of forming a secondary structure, the GC content, the length limit of the amplification product, etc., and screens out the RAA primer that can be used for the rapid detection of HCV. It has good specificity and high sensitivity (minimum detectable 10copies/ ⁇ L).
  • the present invention organically combines colloidal gold lateral flow chromatography test paper technology (Lateral Flow Assay, LFA), which can quickly realize the visual judgment of HCV detection results without complicated professional background.
  • LFA Lateral Flow Assay
  • kits of the present invention to carry out HCV detection mainly includes two steps of isothermal amplification and test strip interpretation, the amplification part takes 10 minutes, and the result interpretation part takes 3 to 5 minutes, compared with the traditional RT-PCR method
  • the shortening of nearly 3 hours can meet the needs of on-site rapid and high-throughput screening, and the detection process is convenient and fast, and the detection results are safe and reliable.
  • Fig. 1 is a schematic diagram of the detection process of hepatitis C virus in the embodiment of the present invention.
  • Fig. 2 is a diagram showing the results of a sensitivity experiment based on the RAA-LFA detection method in the embodiment of the present invention.
  • Fig. 3 is a graph showing the specificity experimental results based on the RAA-LFA detection method in the embodiment of the present invention.
  • RAA amplification lies in the design of primers, but RAA is different from conventional PCR reactions.
  • the present invention selects the C/E genes of HCV 1b genotype strains included in NCBI and other 6 genotype strains for sequence comparison, and carefully selects the conserved region as the RRA amplification target Comprehensive consideration of various factors such as: 1. GC content (40-60%); 2. length (30-35nt); 3. size of the amplification product (100-300bp) and so on.
  • Primer design includes upstream primers and downstream primers. According to the designed upstream RAA primers and downstream RAA primers, cross-matching was carried out, and the best primer pairing was screened to obtain the optimal primers. See Table 1 for details.
  • the specificity and amplification efficiency were further evaluated, and a more preferred primer pair was screened out, specifically: after RAA amplification was performed using the RAA primer pair designed in Table 1, agarose gel electrophoresis and lateral flow chromatography were used to Detection and screening showed that the F2/R2 primer pair had the best amplification effect, good band specificity, and no non-specific amplification. However, other primer pairs have problems such as non-specific amplification and low amplification efficiency of the primers. Therefore, the primer pair F2/R2 was selected for subsequent RAA reaction condition optimization, specificity and sensitivity tests.
  • the specific nucleotide sequence of the more preferred primer pair F2/R2 is: upstream primer: 5'-FITC-CTTGGGATATGATGATGAACTGGTCACCTAC-3' (SEQ ID NO.1); downstream primer: 5'-Biotin-AAGAGTAGCATCACAATCAGAACCTTAGCC-3' (SEQ ID NO.2).
  • the RAA reaction time and reaction temperature were optimized, and it was found that the amplification efficiency of RAA was the highest when the reaction temperature was 37°C for 5 minutes.
  • the optimized RAA reaction condition of the present invention only needs one temperature in the entire gene amplification process, does not require special instruments and equipment, and is simpler to operate, and is suitable for on-site rapid detection of hepatitis C virus.
  • Embodiment two sensitivity and specificity test
  • HAV hepatitis A
  • HBV hepatitis B
  • HAV hepatitis B
  • HIV HIV
  • Syphilis syphilis
  • HPV human papillomavirus
  • RNA Take 2 mL of isolated patient's peripheral blood, centrifuge at 3000 rpm for 5 min, take 200 ⁇ L of supernatant, add it to 560 ⁇ L of AVL lysate containing 5.6 ⁇ g of Carrier RNA, and extract according to the instructions of QIAamp Viral RNA Mini Handbook (Qiagen Company, catalog#52904/52906) For viral RNA, the elution volume is 50 ⁇ L.
  • the RAA kit add 41.5 ⁇ L of buffer solution, 10 ⁇ L of deionized water, 2.5 ⁇ L of upstream primer and downstream primer, 2 ⁇ L of template positive plasmid, and finally add 2.5 ⁇ L of 280 mmol /L magnesium acetate solution, fully mix the RAA amplification system, put it on a water bath, and react at 30-42°C for 5-30min, and obtain the RAA amplification product after the reaction.
  • the amplified product is directly dropped on the colloidal gold-labeled lateral flow chromatography test strip, and the resulting bands are clear.
  • the detection line (T line) of the positive sample is red that can be distinguished by the naked eye, and the negative sample does not develop color (T line).
  • the control line (C line) indicates that the test result is valid.
  • Embodiment 4 Verification test applied to batch detection of patients
  • the RAA kit add 41.5 ⁇ L of buffer solution, 10 ⁇ L of deionized water, 2.5 ⁇ L of upstream primer and downstream primer, 2 ⁇ L of template positive plasmid, and finally add 2.5 ⁇ L of 280 mmol /L magnesium acetate solution, fully mix the RAA amplification system, put it on a water bath, and react at 30-42°C for 5-30min, and obtain the RAA amplification product after the reaction.
  • the amplified product is directly dropped on the colloidal gold-labeled lateral flow chromatography test strip, and the resulting bands are clear.
  • the detection line (T line) of the positive sample is red that can be distinguished by the naked eye, and the negative sample does not develop color (T line).
  • the control line (C line) indicates that the test result is valid.

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Abstract

本发明公开了一种用于检测丙型肝炎病毒的RAA引物、试剂盒及其应用。本发明筛选得到的HCV检测RAA引物特异性好、灵敏度高(最低可检测到10copies/μL)，能够用于制备HCV检测试剂盒及构建RAA扩增体系，并结合侧向流动层析技术实现对HCV快速、低成本的检测及可视化的结果判定，无需复杂的专业背景，使用过程方便快捷，检测结果安全可靠。

Description

用于检测丙型肝炎病毒的RAA引物、试剂盒及其应用
优先权信息
本申请要求于2021年11月25日提交的申请号为202111415246.1的中国申请的优先权，其全部内容通过引用的方式并入本文。
技术领域
本发明涉及病毒生物学检测技术领域，具体涉及一种用于检测丙型肝炎病毒的RAA引物、试剂盒及其应用。
背景技术
丙型肝炎是一种由丙型肝炎病毒(HCV)引起，经血液传播的疾病，是《中华人民共和国传染病防治法》规定的乙类传染病。科技、卫生健康等部门将丙肝相关研究纳入国家科技计划范畴，强化基础性和应用性研究，支持开展新型预防与治疗技术策略研究，快速检测技术的研发与投入，为全面推进健康中国建设、保护人民群众健康奠定基础。
现阶段对丙肝的常规诊断方法为qPCR、PCR与ELISA，三者虽属于成熟的检测方式，但均需要昂贵设备、检测周期较长(3～4h)、试剂耗材成本高，且目前只能在部分市级疾控中心和三甲医疗机构中开展，检测能力不能满足实际需求，急需一种能在普通实验室操作、无需高档设备、可实现快速检测、直接判读的核酸检测方法。
基于不同原理开发的能够在恒定温度下对DNA模板进行高效扩增的技术，称为等温扩增技术(isothermal amplification)。近年来，核酸等温扩增技术发展迅速，它摆脱了传统的温度循环仪，可以在短时间内迅速进行扩增反应，其中重组酶聚合酶等温扩增(recombinase polymerase amplification，RPA；Recombinase Aided Amplification，RAA)技术显示出了自己多方面的优势，具体体现为：能够在37～42℃恒温条件下，30min内完成对DNA的扩增，具有反应迅速、操作简单等技术优点，同时还具有高度特异性和敏感性的特点，而且对硬件设备的要求很低，因此被广泛应用于疾病的诊断与防治，特别适合用于体外诊断、兽医、食品安全、生物安全、农业等领域。
而RPA/RAA的扩增引物则是整个反应的关键所在，但目前尚无用于引物设计的软件或成熟的设计原则，目前也没有大量的数据为其引物设计提供依据，更没有成熟的经验可供遵循，因而找到适用于丙型肝炎病毒的特异性RPA/RAA引物成为当务之急。
公开于该背景技术部分的信息仅仅旨在加深对本发明总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成本领域技术人员所公知的现有技术。
发明内容
本发明的目的在于提供一种用于检测丙型肝炎病毒的RAA引物及试剂盒，以解决现有技术难以快速检测HCV的技术问题；并将其与侧向层析技术相结合，以解决HCV测试成 本高、HCV检测结果难以快速直观的呈现的难题。
为解决上述技术问题，本发明采用如下技术方案：
筛选得到一种用于检测丙型肝炎病毒的RAA引物，其核苷酸序列如下：
上游引物：5’-FITC-CTTGGGATATGATGATGAACTGGTCACCTAC-3’(如SEQ ID NO.1所示)；
下游引物：5’-Biotin-AAGAGTAGCATCACAATCAGAACCTTAGCC-3’(如SEQ ID NO.2所示)。
在一些实施例中，还包括如下核苷酸序列：
上游引物：5’-FITC-AGTACAGGACTGCAATTGCTCAATATATCC-3’(SEQ ID NO.3)；
下游引物：5’-Biotin-GCAAAGATAGCATCACAATCAGAACCTTAG-3’(SEQ ID NO.4)。
所述RAA引物可应用于制备丙型肝炎病毒检测相关产品中，如：
制备一种HCV检测试剂盒，包括RAA引物，以及RNA提取试剂、RAA扩增反应试剂、侧向流动层析试纸条、阳性对照品、阴性对照品等。
构建一种用于HCV检测的RAA扩增体系，包括：
41.5μL缓冲液、去离子水10μL、所述上游引物和下游引物各2.5μL、待测样品RNA提取物2μL及280mmol/L醋酸镁溶液2.5μL。
所述HCV检测试剂盒的使用方法，包括如下步骤：
(1)提取待测样品的丙型肝炎病毒的RNA；
(2)制备阳性标准品，获得阳性质粒；
(3)使用重组酶等温扩增金标检测卡(recombinase-aided amplification and lateral flow assay，RAA-LFA)检测丙型肝炎病毒。
具体而言，是以RNA提取试剂提取待测样品的RNA，利用所述RAA引物及对应的RAA扩增反应试剂进行等温扩增反应，获得RAA扩增产物。
所述等温扩增反应为：向装有反应干粉的0.2mL检测单元管中加入41.5μL缓冲液、去离子水10μL、上游引物和下游引物各2.5μL、模板阳性质粒2μL，最后加入280mmol/L醋酸镁溶液2.5μL，将RAA扩增体系充分混匀，置于水浴锅上，30～42℃反应5～30min，反应结束后获得RAA扩增产物。
同时设置阳性(阳性对照采用阳性质粒pUC57-pC/E1为模板)对照和阴性对照(阴性对照采用不含丙肝病毒基因组片段的空载体质粒样品)。
进一步的，将所得RAA扩增产物滴加于胶体金标记侧向层析试纸条的样品垫上，待反应3～10分钟后，观察试纸条上检测线和质控线通过检测线和质控线的出现情况对检测结果进行判断。
优选的，可先将所述RAA扩增产物于37℃下温育5min后，再用200μL PBST缓冲液稀释，并将其滴加至胶体金标记侧向层析试纸条样本孔中，室温温育5～10min后观察结果。检测线呈现红色，则为阳性(Positive Readout)，检测线不显色，则为阴性(Negative Readout)。
与现有技术相比，本发明的主要有益技术效果在于：
1.本发明综合考虑了靶标区域情况、RAA引物的长度及其形成二级结构的可能性、GC含量、扩增产物的长度限制等诸多因素，筛选得到能用于HCV快速检测的RAA引物，其特异性好、灵敏度高(最低可检测到10copies/μL)。
2.本发明在RPA/RAA基础上有机结合胶体金侧流层析试纸技术(Lateral FlowAssay，LFA)，能够快速实现对HCV检测结果的可视化判定，无需复杂的专业背景。
3.利用本发明试剂盒进行HCV检测主要包括等温扩增与试纸条判读两个步骤构成，扩增部分时间为10分钟，结果判读部分时间为3～5分钟，较传统的RT-PCR方法缩短近3小时，能满足现场快速、高通量筛查的需求，且检测过程方便快捷，检测结果安全可靠。
附图说明
图1为本发明实施例中丙型肝炎病毒检测流程示意图。
图2为本发明实施例中基于RAA-LFA检测方法所示的敏感性实验结果图。
图3为本发明实施例中基于RAA-LFA检测方法所示的特异性实验结果图。
具体实施方式
下面结合附图和实施例来说明本发明的具体实施方式，但以下实施例只是用来详细说明本发明，并不以任何方式限制本发明的范围。
另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其它陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本说明书中使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本说明书所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
实施例一：引物设计与条件优化
RAA扩增的关键在于引物的设计，但RAA不同于常规的PCR反应，目前尚无用于引物设计的软件或成熟的设计原则，也没有大量的数据为其引物设计提供依据，更没有成熟的经验可供遵循；基于长期的实践研究，本发明选取NCBI收录的HCV 1b基因型毒株及其它6个基因型毒株的C/E基因进行序列比对，并审慎选取保守区域作为RRA扩增靶标；综合考虑多种因素如：1.GC含量(40～60％)；2.长度(30～35nt)；3.扩增产物大小(100～300bp)等等。引物设计包括上游引物和下游引物。根据设计的上游RAA引物及下游RAA引物，进行了交叉配对，并对最佳引物配对做了筛选，获得最优引物。具体见表1。
表1.针对丙型肝炎病毒C/E1基因的保守区设计的RAA引物。
Figure PCTCN2022126394-appb-000001
进一步对特异性和扩增效率进行评估，筛选出更优选的引物对，具体为：采用表1中所设计的RAA引物对进行RAA扩增后，通过琼脂糖凝胶电泳和侧向层析技术进行检测筛选得到F2/R2引物对的扩增效果最好，条带特异性好，没有非特异性扩增。而其它的引物对出 现非特异性扩增、引物的扩增效率低等问题。因此，选择引物对F2/R2进行后续的RAA反应条件优化、特异性和敏感性试验。更优选的引物对F2/R2的具体核苷酸序列为：上游引物：5’-FITC-CTTGGGATATGATGATGAACTGGTCACCTAC-3’(SEQ ID NO.1)；下游引物：5’-Biotin-AAGAGTAGCATCACAATCAGAACCTTAGCC-3’(SEQ ID NO.2)。
对RAA反应时间和反应温度进行优化，结果发现，反应温度为37℃，作用5min时，RAA扩增效率最高。本发明优化后的RAA反应条件，其整个基因扩增过程只需一个温度，不需要特殊的仪器设备，操作更简单，适用丙型肝炎病毒的现场快速检测。
实施例二：敏感性和特异性试验
选择标准丙型肝炎病毒质粒样品为模板，双蒸水10倍梯度稀释浓度为10  8copies/μL-1copies/μL的标准质粒模板，并以双蒸水作为空白对照(CK)，按实施例一中所记载优化后的反应条件进行RAA检测。结果为最低检出模板量为10copies/μL(见图2)；灵敏度较常规PCR方法高，且本发明的检测方法更为简单、快速，尤其适于现场检测，对实验条件的要求更宽松。
分别用传染性六项疾病中的其它五项甲肝(HAV)、乙肝(HBV)、艾滋(HIV)、梅毒(Syphilis)、人乳头瘤病毒(HPV)阳性样本提取核酸样本为模板，按实施例一中优化后的反应条件进行RAA，通过凝胶电泳和侧向层析技术进行检测。
侧向层析技术的实验结果如图3所示，只有丙型肝炎病毒(HCV)为阳性，其它的均为阴性，说明本发明的RAA检测体系特异性良好。
实施例三：应用于患者个体筛查的验证试验
取离体的患者外周血2mL，3000rpm离心5min，取200μL上清，加入含有5.6μg Carrier RNA的560μL的AVL裂解液中，按QIAamp Viral RNA Mini Handbook(Qiagen公司，catalog#52904/52906)说明书提取病毒RNA，洗脱体积50μL。按照RAA试剂盒操作说明进行，向装有反应干粉的0.2mL检测单元管中加入41.5μL缓冲液、去离子水10μL、上游引物和下游引物各2.5μL、模板阳性质粒2μL，最后加入2.5μL 280mmol/L的醋酸镁溶液，将RAA扩增体系充分混匀，置于水浴锅上，30～42℃反应5～30min，反应结束后获得RAA扩增产物。将扩增产物直接滴加于胶体金标记侧向层析试纸条，产生的条带清晰，阳性标本检测线(T线)呈现肉眼可以分辨的红色，阴性标本不显色(T线)，控制线(C线)表明检测结果有效。
实施例四：应用于患者批量检测的验证试验
取离体的患者外周血2mL，3000rpm离心5min，取200μL上清。使用全自动核酸提取仪批量提取患者血清中的病毒RNA，洗脱体积50μL。按照RAA试剂盒操作说明进行，向装有反应干粉的0.2mL检测单元管中加入41.5μL缓冲液、去离子水10μL、上游引物和下游引物各2.5μL、模板阳性质粒2μL，最后加入2.5μL 280mmol/L的醋酸镁溶液，将RAA扩增体系充分混匀，置于水浴锅上，30～42℃反应5～30min，反应结束后获得RAA扩增产物。将扩增产物直接滴加于胶体金标记侧向层析试纸条，产生的条带清晰，阳性标本检测线(T线)呈现肉眼可以分辨的红色，阴性标本不显色(T线)，控制线(C线)表明检测结果有效。
上面结合附图和实施例对本发明作了详细的说明，但是，所属技术领域的技术人员能够理解，在不脱离本发明构思的前提下，所作的改变、修饰、替代、组合、简化，均应为等效的替代方式，从而形成多个具体的实施例，均为本发明的常见变化范围。

Claims (10)

  1. 一种用于检测丙型肝炎病毒的RAA引物，其特征在于，其核苷酸序列如下：
    上游引物：5’-FITC-CTTGGGATATGATGATGAACTGGTCACCTAC-3’(SEQ ID NO.1)；
    下游引物：5’-Biotin-AAGAGTAGCATCACAATCAGAACCTTAGCC-3’(SEQ ID NO.2)。
  2. 根据权利要求1所述的RAA引物，其特征在于，还包括如下核苷酸序列：
    上游引物：5’-FITC-AGTACAGGACTGCAATTGCTCAATATATCC-3’(SEQ ID NO.3)；
    下游引物：5’-Biotin-GCAAAGATAGCATCACAATCAGAACCTTAG-3’(SEQ ID NO.4)。
  3. 一种丙型肝炎病毒检测试剂盒，其特征在于，包括权利要求1或2所述RAA引物。
  4. 根据权利要求3所述的试剂盒，其特征在于，还包括RNA提取试剂、RAA扩增反应试剂、侧向流动层析试纸条、阳性对照品、阴性对照品。
  5. 一种用于检测丙型肝炎病毒的RAA扩增体系，其特征在于，包括：
    41.5μL缓冲液、去离子水10μL、权利要求1或2所述上游引物和下游引物各2.5μL、待测样品RNA提取物2μL及280mmol/L醋酸镁溶液2.5μL。
  6. 权利要求3或4所述试剂盒的使用方法，其特征在于，包括如下步骤：
    以RNA提取试剂提取待测样品的RNA，利用所述RAA引物及对应的RAA扩增反应试剂进行等温扩增反应，获得RAA扩增产物。
  7. 根据权利要求6所述的使用方法，其特征在于，所述等温扩增反应为：向装有反应干粉的0.2mL检测单元管中加入41.5μL缓冲液、去离子水10μL、上游引物和下游引物各2.5μL、模板阳性质粒2μL，最后加入280mmol/L醋酸镁溶液2.5μL，将RAA扩增体系充分混匀，置于水浴锅上，30～42℃反应5～30min，反应结束后获得RAA扩增产物。
  8. 根据权利要求7所述的使用方法，其特征在于，还包括如下步骤：将所得RAA扩增产物滴加于胶体金标记侧向层析试纸条的样品垫上，待反应3～10分钟后，观察试纸条上检测线和质控线通过检测线和质控线的出现情况对检测结果进行判断。
  9. 根据权利要求8所述的使用方法，其特征在于，先将所述RAA扩增产物于37℃下温育5min后，再用200μL PBST缓冲液稀释，并将其滴加至胶体金标记侧向层析试纸条样本孔中，室温温育5～10min后观察结果。
  10. 权利要求1或2所述RAA引物在制备丙型肝炎病毒检测相关产品中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114381551A (zh) * 2021-12-21 2022-04-22 佛山科学技术学院 一种用于检测大口黑鲈虹彩病毒的实时荧光raa引物、探针及试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862262B (zh) * 2021-11-25 2023-10-17 郑州大学 用于检测丙型肝炎病毒的raa引物、试剂盒及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070790B1 (en) * 1993-06-29 2006-07-04 The United States Of America As Represented By The Department Of Health And Human Services Nucleotide and deduced amino acid sequences of the envelope 1 and core genes of isolates of hepatitis C virus and the use of reagents derived from these sequences in diagnostic methods and vaccines
CN112501357A (zh) * 2020-12-22 2021-03-16 许晏铭 一种基于rpa-lfd法检测丙肝病毒的rpa引物及其应用
CN113862262A (zh) * 2021-11-25 2021-12-31 郑州大学 用于检测丙型肝炎病毒的raa引物、试剂盒及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8900850B2 (en) * 2009-09-17 2014-12-02 Michael J. Lane Lateral flow based methods and assays for rapid and inexpensive diagnostic tests
CN107541571B (zh) * 2017-10-20 2018-09-28 中国农业科学院兰州兽医研究所 Rt-rpa与侧向流动层析技术相结合的猪嵴病毒快速检测方法及其检测试剂盒
CN108841926B (zh) * 2018-07-13 2021-10-01 锦州医科大学 一种rt-rpa-侧流层析双重检测戊型肝炎病毒和甲型肝炎病毒的引物、探针及试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070790B1 (en) * 1993-06-29 2006-07-04 The United States Of America As Represented By The Department Of Health And Human Services Nucleotide and deduced amino acid sequences of the envelope 1 and core genes of isolates of hepatitis C virus and the use of reagents derived from these sequences in diagnostic methods and vaccines
CN112501357A (zh) * 2020-12-22 2021-03-16 许晏铭 一种基于rpa-lfd法检测丙肝病毒的rpa引物及其应用
CN113862262A (zh) * 2021-11-25 2021-12-31 郑州大学 用于检测丙型肝炎病毒的raa引物、试剂盒及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CORBET SYLVIE, BUKH JENS, HEINSEN ANJA, FOMSGAARD ANDERS: "Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 41, no. 3, 1 March 2003 (2003-03-01), US , pages 1091 - 1100, XP093069192, ISSN: 0095-1137, DOI: 10.1128/JCM.41.3.1091-1100.2003 *
ZHOU SUJUAN, ZHANG TIE-JUN, HE NA: "Methodological Improvement for PCR Amplification of the C/E1 Region in HCV Genome", CHINESE JOURNAL OF DISEASE CONTROL & PREVENTION, vol. 14, no. 8, 10 August 2010 (2010-08-10), pages 736 - 738, XP093069191, ISSN: 1674-3679 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114381551A (zh) * 2021-12-21 2022-04-22 佛山科学技术学院 一种用于检测大口黑鲈虹彩病毒的实时荧光raa引物、探针及试剂盒
CN114381551B (zh) * 2021-12-21 2024-02-02 佛山科学技术学院 一种用于检测大口黑鲈虹彩病毒的实时荧光raa引物、探针及试剂盒

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