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 palin-dromic 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 opti-mized to establish the RAA-CRISPR/Cas13a-LFA detection system.The sensitivity and specificity of the sys-tem 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(MeV).After evaluation with clinical samples,no significant difference was found between the RAA-CRISPR/Cas13a-LFA and qPCR methods(P＞0.05),and they exhib-ited 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％,re-spectively.Conclusion The RAA-CRISPR/Cas13a-LFA detection system can quickly,sensitively,and specifically detect MPXV.
维普
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