A RAPID CRISPR/CAS13A SYSTEM-BASED METHOD FOR DETECTING SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS
Objective To develop a rapid and sensitive on-site nucleic acid detection method based on the clustered regularly interspaced short palindromic repeats(CRISPR)system to detect severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Firstly,a specific conserved region of the SFTSV S gene was identified by sequence alignment analysis,followed by the design and selection of multienzyme isothermal rapid amplification(MIRA)primers and target CRISPR RNA(crRNA);detection results were read using fluorescence signals and immunochromatographic paper.Finally,in order to increase sensitivity,multiple detection targets of crRNA were incorporated into a single reaction system.The effectiveness of this method for field nucleic acid detection in tick samples was validated using simulated tick-infected samples and field ticks.Results The minimum detection limit(MDL)of the MIRA-CRISPR/Cas13a fluorescence assay for SFTSV was 1 copy/μL,while that of the stripe-based method was 10 copies/μL.No cross-reactivity was observed between SFTSV and the other four viral controls,indicating the high specificity of the MIRA-CRISPR/Cas13a system.The method demonstrated superior sensitivity for nucleic acid detection in simulated tick infection samples compared to RT-PCR.The analysis of 29 field tick samples revealed that 20 and 9 samples were positive and negative,respectively(100%agreement with RT-PCR results).Conclusions The proposed method could facilitate the rapid detection of SFTSV in the field.
国家科技期刊平台
NSTL
万方数据
