猴痘病毒数字PCR检测方法的建立

Establishment of digital PCR method for detection of monkeypox virus

目的建立猴痘病毒(MPXV)通用型、西非分支、中非分支的数字PCR检测方法,为口岸猴痘病毒精准防控提供技术支持。方法比对分析猴痘病毒不同分支及其他正痘病毒科成员如牛痘病毒、痘苗病毒等序列,以猴痘病毒G2R基因、C3L基因为靶基因设计3组特异性引物和荧光探针序列,分别构建猴痘病毒通用型(G2R_GR)、西非分支(G2R_WA)和中非分支(C3L_CA)数字PCR检测方法。结果构建的猴痘病毒数字PCR通用型检测方法对西非分支、中非分支均能检出,对其他正痘病毒科成员如牛痘、痘苗等均未检出,西非分支(G2R_WA)、中非分支(C3L_CA)均具有较好的检测特异性。数字PCR方法检测灵敏度较高,检测限为45拷贝/ml。结论构建的数字PCR方法可实现对猴痘病毒通用型、西非分支和中非分支的特异性检测,为猴痘病毒口岸防控提供有力技术支持。

Objective To establish digital PCR detection method for monkeypox virus(MPXV)including universal type,West African branch and Central African branch,provide technical support for the precise prevention and control of MPXV at ports.Methods By comparing and analyzing the sequences of different branches of MPXV and other members of orthopoxvirus family,such as cowpox and vaccinia virus,3 sets of specific primers and probes were designed targeted MPXV G2R and C3L genes,and digital PCR methods were constructed to detect MPXV of universal type(G2R_GR),West African branch(G2R_WA)and Central African branch(C3L_CA),respectively.Results The established universal digital PCR detection method for MPXV could detect both West African branch and Central African branch,but could not detect other members of orthopoxvirus family such as cowpox and vccinia virus.Both the West African branch(G2R_WA)and Central African branch(C3L_CA)had good specifiaty.The detection sensitivity of the digital PCR method was higher,reached 45 copies/ml.Conclusion The digital PCR detection method constructed could achieve specific detection of MPXV of universal,West African branch and Central African branch,provided strong technical support for the prevention and control of MPXV at ports.