猴痘病毒一步法MIRA-Crispr/cas12a快速检测方法的建立

Establishment of one-step MIRA-Crispr/cas12a rapid detection method for monkeypox virus

目的 建立快速、特异、灵敏、便携的猴痘病毒(MPXV)多酶恒温快速扩增(MIRA)-Crispr/cas12a一步检测法。方法 选取MPXV F3L和G2Rcom、刚果分支(Ⅰ型)D14L和西非分支(Ⅱ型)G2Rcom基因为靶标,设计合成引物、crRNA及阳性质粒。基于含MPXV F3L和G2Rcom基因的阳性质粒,设置10^(6)、10^(4)、10^(3)、10^(2)拷贝/ml共4个浓度,利用MIRACrispr/cas12a技术建立荧光检测法并评价其灵敏性;以痘苗病毒、牛痘病毒、羊痘病毒、鼠痘病毒作为对照评价特异性;利用阳性质粒模拟临床样本验证建立的方法。基于Ⅰ型D14L和Ⅱ型G2Rcom基因的阳性质粒,建立Ⅰ型和Ⅱ型的MIRA-Crispr/cas12a荧光检测法,并在4种对照病毒的基础上,选用Ⅱ型或Ⅰ型作为对照进行特异性评价。建立MIRA-Crispr/cas12a可视化检测方法,利用侧向流试纸条直接判读结果。结果 MPXV F3L和G2Rcom、Ⅰ型D14L和Ⅱ型G2Rcom基因均得到特异性扩增。基于MPXV F3L和G2Rcom基因的方法灵敏性均为10^(3)拷贝/ml,灵敏性和特异性均较好;基于Ⅰ型D14L和Ⅱ型G2Rcom基因的方法特异性较好。模拟临床样本检测发现,基于MPXV F3L和G2Rcom基因的方法检出6份阳性模拟样本和2份阴性健康人咽拭子,与实时荧光PCR法的结果一致。基于MPXV F3L和G2Rcom、Ⅰ型D14L和Ⅱ型G2Rcom基因的可视化检测,在106拷贝/ml浓度时均显示两条带,10^(8)拷贝/ml均显示一条带。结论 建立的检测MPXV方法操作简单,一次开盖加样,灵敏性和特异性较好,可在30 min内完成检测,实现“样品进,结果出”,适用于MPXV的快速检测。

Objective To establish a rapid,specific,sensitive and portable method for the detection of monkeypox virus(MPXV)by multienzyme isothermal rapid amplification(MIRA)-Crispr/cas12a.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 10^(6),10^(4),10^(3)and 10^(2)copies/ml were set.MIRA-Crispr/cas12a fluorescence methods 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 10^(3) copies/ml,with good sensitivity 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 detection 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 10^(8) 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.