非洲猪瘟病毒一步法巢式锁核酸荧光定量PCR方法的建立  

Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus

作　　者：施科达 徐民生 李艳 张昆丽 翟少伦 勾红潮 宋帅 杨冬霞 臧莹安 孙铭飞 李春玲 SHI Keda;XU Minsheng;LI Yan;ZHANG Kunli;ZHAI Shaolun;GOU Hongchao;SONG Shuai;YANG Dongxia;ZANG Ying'an;SUN Mingfei;LI Chunling(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention of Guangdong Province/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture and Rural Affairs,P.R.China,Guangzhou 510640;Maoming Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology/College of Animal Science&Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广州510640 [2]岭南现代农业科学与技术广东省实验室茂名分中心/仲恺农业工程学院动物科技学院,广州510225

出　　处：《中国农学通报》2023年第11期143-151,共9页Chinese Agricultural Science Bulletin

基　　金：广东省重点领域研发计划资助(2019B020211005,2019B020217002);广东省现代农业产业技术体系创新团队项目(2022KJ119);广州市科技特派员项目(20212100027);广东省农业科学院院长基金项目(202044)。

摘　　要：试验基于非洲猪瘟病毒(African swine fever virus,ASFV)B646L(P72)蛋白基因序列,建立一种高灵敏度和高特异性的检测方法。采用Oligo 7软件设计一步法巢式锁核酸荧光定量PCR(One Step NestedLocked Nucleic Acid real-time PCR,LNA-OSN-PCR)的嵌套引物以及探针,并对外引物进行锁核酸修饰。建立并优化LNA-OSN-PCR反应体系和条件,确立LNA-OSN-PCR标准曲线,并检验LNA-OSN-PCR方法的灵敏度、特异性和重复性。采用LNA-OSN-PCR方法,对96份临床疑似患病样品进行检测,同时与普通荧光探针PCR方法进行比较。试验确立并优化了LNA-OSN-PCR的反应条件和体系,建立的LNA-OSN-PCR标准曲线,具有较好的线性(R2=0.9945)。该方法的最低检测限为3×10^(-1)copies/μL,变异系数小于3%,并且与其他病毒或细菌核酸无交叉反应。LNA-OSN-PCR方法与OIE推荐的荧光定量PCR方法比较,检测灵敏度提高10~100倍,在对96份临床疑似患病核酸样品进行检测中,检出55份阳性,41份阴性,比常规的荧光定量PCR具有更高的阳性检出率。并且能获得典型的扩增曲线。试验结合一步法巢式PCR和锁核酸修饰,建立了ASFV一步法巢式锁核酸荧光定量PCR,为检测ASFV提供一种高灵敏度和高特异性且经济实惠的检测方法。In this study,a highly sensitive and specific detection method was established based on the B646L(P72)protein gene of African swine fever virus(ASFV).The nested primers and probes of LNA-OSN-PCR(One Step Nested-Locked Nucleic Acid real-time PCR)were designed with Oligo 7 software;external primers were modified with locked nucleic acids.The reaction system and conditions of LNA-OSN-PCR were established and optimized,and the standard curve of LNA-OSN-PCR was established.Then,the sensitivity,specificity and repeatability of LNA-OSN-PCR were tested.A total of 96 clinical suspected diseased samples were detected by LNA-OSN-PCR and compared with the samples detected by conventional fluorescent PCR method.The reaction conditions and system of LNA-OSN-PCR were established and optimized,and the standard curve of LNA-OSN-PCR had good linearity(R2=0.9945).The LNA-OSN-PCR had a minimum detection limit of 3×10^(−1) copies/μL,with a coefficient of variation of less than 3%and no cross-reaction with other viral or bacterial nucleic acids.Compared with the qPCR method recommended by OIE,the detection sensitivity of the LNA-OSN-PCR method was increased by 10-100 times.Among the 96 samples tested by LNA-OSN-PCR,55 were positive and 41 were negative,with a higher positive detection rate than traditional qPCR and a better typical amplification curve.By combining the one-step nested PCR and lock-in nucleic acid modification,the LNA-OSN-PCR method for ASFV was established to provide a highly sensitive,specific and economical method for ASFV detection.

关 键 词：非洲猪瘟病毒 B646L(P72)蛋白基因 锁核酸修饰 一步法巢式荧光定量PCR 

分 类 号：S854.4[农业科学—临床兽医学]