摘要
【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.
作者
曾宇晨
龚浪
王京煜
潘昊鸣
梁杏玲
马俊
张桂红
王衡
ZENG Yuchen;GONG Lang;WANG Jingyu;PAN Haoming;LIANG Xingling;MA Jun;ZHANG Guihong;WANG Heng(College of Veterinary medicine,South China Agricultural University/Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province,Guangzhou 510642,China;African Swine Fever Regional Laboratory of China(Guangzhou)/Prevention and Control Technology Research Center for African Swine Fever,South China Agricultural University,Guangzhou 510642,China;National Engineering Research Center for Breeding Swine Industry,South China Agricultural University,Guangzhou 510642,China)
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2021年第5期33-40,共8页
Journal of South China Agricultural University
基金
广东省重点领域研发计划(2019B020211005,2019B020211003)
国家自然科学基金(31941004)
财政部和农业农村部国家现代农业技术产业体系基金(CARS-35)。
关键词
非洲猪瘟
酶促重组酶扩增
等温扩增
快速检测
African swine fever
enzymatic recombinase amplification
isothermal amplification
rapid detection
