基于B646L、EP402R、MGF360/505基因的非洲猪瘟病毒ERA检测方法的建立

Development of ERA detection method for African swine fever virus based on B646L,EP402R and MGF360/505 genes

【目的】建立一种基于酶促重组酶扩增(Enzymatic recombinase amplification,ERA)技术的非洲猪瘟病毒(African swine fever virus,ASFV)DNA快速检测方法。【方法】参考ASFV B646L基因、EP402R基因和多基因家族成员MGF360/505基因保守序列,设计特异性的ERA探针和引物,经过反应条件的优化,建立42℃等温条件下检测ASFV DNA的ERA方法。【结果】ERA-ASFV-B646L、ERA-ASFV-EP402R和ERA-ASFV-MGF3个检测方法分别在16、7和13 min内即可得出检测结果;特异性强,对阳性样品拷贝数的检测下限均为102μL-1;与我国非洲猪瘟诊断技术标准中的方法对比,结果符合率为100%。【结论】本研究建立的ERA检测方法可以用于ASFV的快速检测,为流行病学调查和现场检测提供了一种快速有效的检测方法。

【Objective】To establish a rapid detection method of African swine fever virus(ASFV)DNA based on enzymatic recombinase amplification(ERA)technology.【Method】Specific ERA probes and primers were designed according to the conserved sequences of B646 L and EP402 R genes,and MGF360/505 gene which was one of the ASFV multigene family members.Through optimizing reaction conditions,the ERA method for detecting ASFV DNA was finally established under the isothermal condition of 42℃.【Result】The detection results of three methods of ERA-ASFV-B646 L,ERA-ASFV-EP402 R and ERA-ASFV-MGF could be obtained within 16,7 and 13 min respectively.The copy number detection limitation of positive sample was all102μL-1.The coincidence rate was 100%compared with the common method in the technical standard of ASFV diagnosis in China.【Conclusion】The established ERA method can be used for rapid detection of ASFV,and provides an effective rapid detection method for epidemiological investigation and field detection.