• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

RAA-CRISPR/cas13a(cpf1)-LFD兔出血症2型诊断方法的建立

Development of RAA-CRISPR/Cas13a(cpf1)-LFD Technology for Rabbit Hemorrhagic Disease Virus

    摘要
  • 摘要:
    目的 2020年以来，兔出血症2型（rabbit hemorrhagic disease type 2, RHdV-2）在我国被发现，并呈地方性流行。临床上急需一种适用于生产一线，且快速简便不需昂贵仪器的检测方法。
    方法 首先采用生物信息学软件（Vector NTI alignment）分析兔出血症1型（rabbit hemorrhagic disease type 1, RHdV-1）和兔出血症2型RHdV-2基因组，筛选RHdV-2特异且保守的VP60基因片段，设计重组酶介导等温核酸扩增技术-侧流层析试纸条检测（recombinase aided amplification - lateral flow dipstick, RAA-LFD）探针与特异性引物，然后在RAA扩增产物的基础上，设计并筛选最适CRISPR衍生RNA（CRISPR-derived RNA, crRNA），并对簇状规律间隔性成簇短回文重复序列相关蛋白13a（clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 13a, CRISPR-Cas13a）的反应体系进行优化。筛选针对RHdV-2的RAA-CRISPR/Cas13a (cpf1)-LFD诊断方法的最适反应时间，并对该方法的敏感性、特异性进行评价。最后使用该方法对临床31份疑似样品进行检测。
    结果 同源性分析结果表明，RHdV-2的VP60基因上存在特异且保守的序列，合成VP60特异性引物进行RAA扩增。以RAA产物作为反应模板，发现crRNA1特异性最好。反应时间结果表明，在RAA基础反应后仅再需10 min即可在试纸条上出现明显阳性结果。敏感性试验结果显示，以重组质粒为模板最低检出质粒拷贝数为6.7×101 copies·μL−1。特异性结果表明，只有RHdV-2为阳性，表明该方法特异性好。采用RAA-CRISPR/Cas13a(cpf1)-LFD法对31份临床样品进行检测，检出阳性样品10份，检出率为30.3%，说明该方法敏感性好。
    结论 本研究建立了一种针对RHdV-2的快速、简便的RAA-CRISPR/Cas13a (cpf1)-LFD检测方法，该方法对快速确诊RHdV-2以及科学防控该病具有十分重要的意义。

     

    Abstract:
    Objective Since 2020, rabbit hemorrhagic disease virus type 2 (RHdV-2) was isolated and identified in China, and it was endemic. So a quick and easy diagnostic methods is urgently needed.
    Method Initially, the bioinformatics software Vector NTI alignment was utilized to analyze the genomic sequences of two serotypes rabbit hemorrhagic disease type 1 and rabbit hemorrhagic disease type 2, and identify a specific and conserved fragment VP60 of RHdV-2 RAA-LFD probes and specific primers were designed accordingly. Subsequently, based on the RAA amplification products, the optimal CRISPR-derived RNA was designed and screened, and the reaction conditions of RAA-CRISPR/Cas13a (Cpf1)-LFD was optimized. The sensitivity and specificity were evaluated. RAA esteblished in our previous study and RAA-CRISPR/Cas13a(Cpf1)-LFD detection method were applied to the detection of clinical samples.
    Result The results of the homology analysis indicated that there were specific and conserved sequences on the VP60 gene of RHdV-2. Based on this specific and conserved gene fragment, VP60-specific primers were synthesized for RAA amplification. Using the RAA production as the reaction template, the same positive sample was detected, and it was found that crRNA1 had higher specificity. In terms of reaction time, only an additional 10 minutes were needed after the basic RAA reaction to obtain a clear positive result on the test strip. The sensitivity test results showed that the lowest detectable plasmid copy number with the recombinant plasmid as the template was 6.7×101 copies·μL−1, which was 100 times more sensitive than the RAA-LFD method. The specificity results of RAA-CRISPR/Cas13a(Cpf1)-LFD test showed that only RHdV-2 was positive, indicating it had good specificity.The detection of 31 clinical samples using the RAA-CRISPR/Cas13a(cpf1)-LFD method resulted in 10 positive samples, with a detection rate of 25.8%, indicating that the method has good sensitivity.
    Conclusion In this study, we developed RAA-CRISPR/Cas13a(Cpf1)-LFD for RHdV-2, which was quick and easy.The method is of great significance for the rapid diagnosis of RHdV-2 and the scientific prevention and control of the disease.

     

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