重组酶介导等温扩增-CRISPR/Cas13a结合侧流层析技术快速检测猴痘病毒

Rapid detection of monkeypox virus using recombinase-aided amplification-CRISP/Cas13a combined with lateral flow assay

目的基于重组酶介导的等温扩增技术(recombinase aided amplification,RAA)和规则成簇间隔短回文重复序列(cluste redregularly interspaced short palindromic repeats,CRISPR)-Cas13a(CRISPRassociated protein 13a)结合侧流层析技术(lateral flow assay,LFA)建立一种快速、灵敏、特异的猴痘病毒(monkeypox virus,MPXV)检测方法。方法针对MPXV的F3L段基因设计其RAA扩增引物、CRISPR RNA(crRNA)、荧光报告探针及LFA探针,对反应条件进行优化,建立RAA-CRISPR/Cas13a-LFA检测体系,评价该体系的检测灵敏度与特异性,并与实时荧光定量PCR(quantitative real-time PCR,qPCR)检测结果进行一致性比较,同时应用实际临床样本验证该检测体系的准确性。结果RAA-CRISPR/Cas13aLFA检测体系可以在60 min内(包括实验操作)完成检测,该体系对病毒DNA的检出限为18拷贝/反应;特异性试验表明其与水痘-带状疱疹病毒(varicella-zoster virus,VZV)、单纯疱疹病毒(herpes simplex virus,HSV)、EB病毒(Epstein-Barr virus,EBV)、柯萨奇病毒A16型(CVA16)、肠道病毒A组71型(EV-A71)、麻疹病毒(measles virus,MeV)均无交叉反应;临床样本检测结果表明,RAA-CRISPR/Cas13a-LFA与qPCR法之间的差异无统计学意义,二者具有良好的检测一致性(Kappa=0.892)。RAACRISPR/Cas13a-LFA检测体系的灵敏度和特异度分别为100.0%和83.3%,阳性预测值为96.6%,阴性预测值为100.0%。结论建立的RAA-CRISPR/Cas13a-LFA检测体系可快速、灵敏、特异地检测MPXV。

Objective To establish a rapid,sensitive,and specific detection method for monkeypox virus(MPXV)based on recombinase-aided amplification(RAA)and the clustered regularly interspaced short palindromic repeats(CRISPR)-Cas13a(CRISPR associated protein 13a)combined with lateral flow chromatography(LFA)technology.Methods Primers for RAA amplification,CRISPR RNA(crRNA),fluorescence report probes,and LFA probes targeting the F3L gene of MPXV were designed.The reaction conditions were optimized to establish the RAA-CRISPR/Cas13a-LFA detection system.The sensitivity and specificity of the system were evaluated,and the results were compared with quantitative real-time PCR(qPCR).The accuracy of the detection system was further validated using clinical samples.Results The RAA-CRISPR/Cas13a-LFA detection system could complete the detection within 60 minutes(including experimental operations).The limit of detection was 18 copies of virus DNA per reaction.Specificity tests showed no cross-reactivity with varicella-zoster virus(VZV),herpes simplex virus(HSV),Epstein-Barr virus(EBV),coxsackie virus A16(CVA16),human enterovirus 71(EV-A71),or measles virus(Me V).After evaluation with clinical samples,no significant difference was found between the RAA-CRISPR/Cas13a-LFA and q PCR methods(P>0.05),and they exhibited good consistency(Kappa=0.892).The sensibility and specificity of the RAA-CRISPR/Cas13a-LFA were 100.0%and 83.3%,respectively.The positive and negative predictive values were 96.6%and 100.0%,respectively.Conclusion The RAA-CRISPR/Cas13a-LFA detection system can quickly,sensitively,and specifically detect MPXV.