Establishment of one-step MIRA-Crispr/cas12a rapid detection method for monkeypox virus
Objective To establish a rapid,specific,sensitive and portable method for the detection of monkeypox virus(MPXV)by multienzyme isothermal rapid amplification(MIRA)-Crispr/cas 12a.Methods Based on MPXV F3L and G2Rcom,Congo branch(type Ⅰ)D14L,and West Africa branch(type Ⅱ)G2Rcom genes,primers,crRNA and positive plasmids were designed and synthesized.Based on positive plasmids containing MPXV F3L and G2Rcom genes,four concentrations of 106,104,103 and 102 copies/ml were set.MIRA-Crispr/cas12a fluorescence meth-ods were established,the sensitivity and specificity were evaluated by using vaccinia virus,cowpox virus,sheep and goat pox virus and ectromelia virus as controls.The established method was validated by simulating clinical samples with positive plasmids.Based on the positive plasmids of type Ⅰ D14L and type Ⅱ G2Rcom genes,MIRA-Crispr/cas12a fluorescence methods were established,and the specificity were evaluated by using four control viruses and type Ⅱ or type Ⅰ as controls.MIRA-Crispr/cas12a visualization detection methods were built and directly interpreted the results using lateral flow test strips.Results The MPXV F3L and G2Rcom,type Ⅰ D14L and type Ⅱ G2Rcom genes were all specifically amplified.The sensitivity based on MPXV F3L and G2Rcom were both 103 copies/ml,with good sen-sitivity and specificity.The method established based on type Ⅰ D14L and type Ⅱ G2Rcom genes also had good specificity.Simulated clinical samples testing revealed that the methods based on MPXV F3L and G2Rcom genes detected 6 positive simulated samples and 2 negative throat swabs from healthy individuals,consistent with the de-tection results of Real-time fluorescence PCR.Based on visual detection MIRA-Crispr/cas12a methods of MPXV F3L and G2Rcom,type Ⅰ D14L and type Ⅱ G2Rcom genes,two bands were displayed at a concentration of 106 copies/ml,respectively,one band was displayed at a concentration of 108 copies/ml,respectively.Conclusion The detection method for MPXV established in this study was simple in operation,with one open cover and sample addition,good sensitivity and specificity.It could achieve detection within 30 minutes,achieving"sample in,result out"and was suitable for rapid detection of MPXV.
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