胞内劳森菌TaqMan实时荧光定量PCR检测方法的建立及应用  

Development and application of a TaqMan real-time PCR assay for specific detection of Lawsonia intracellularis

作　　者：刘昌锦 魏黄思梧 胡换仪 肖童 林敏[1] 刘小兰[1] 边彦超 罗峰 邓舜洲[1] LIU Changjin;WEI Huangsiwu;HU Huanyi;XIAO Tong;LIN Min;LIU Xiaolan;BIAN Yanchao;LUO Feng;DENG Shunzhou(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;Jiangxi Jinyibo Biotechnology Company,Nanchang 330013,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]江西金伊博生物科技有限公司,江西南昌330013

出　　处：《中国兽医学报》2022年第4期680-685,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31460666);江西省科技支撑计划资助项目(20132BBF60044,20141BBF60039);江西省现代农业产业技术体系建设专项基金资助项目(JXARS-03)。

摘　　要：为建立用于检测临床粪便样品中胞内劳森菌(Lawsonia intracellularis,L.intracellularis)的TaqMan荧光定量PCR方法,本研究参考GenBank中发表的L.intracellularis PHE/MN1-00株基因组序列设计50对引物,通过SYBR GreenⅠ荧光染料法PCR验证引物的特异性,针对扩增效率最好的特异性上、下游引物合成相应的TaqMan探针,优化反应条件,对其线性范围、敏感性和重复性进行验证,系统的完成了该方法的建立。结果表明,所建立的TaqMan荧光定量PCR方法,特异性强;在靶标核酸为2.95×10^(1)~2.95×10^(6)拷贝/μL区间线性关系良好,相关系数R^(2)=0.9981,扩增效率为96.51%;最低检测浓度为5拷贝/μL;批间和批内的CV均小于2%,重复性好。使用普通PCR方法与本方法对2020年10月至2021年2月采自江西地区猪场的373份粪便样品进行检测,L.intracellularis的总检出率分别为21.4%(80/373)和27.6%(103/373),显示本方法具有更高的敏感性,其检测场阳性率为88.9%(8/9),各阶段生产猪群阳性率分别为经产母猪28.7%、保育仔猪36.4%和育肥猪14.3%。该检测结果为江西地区的L.intracellularis的流行情况提供参考。The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis(Lawsonia intracellularis,L.intracellularis)in porcine feces.In this study,50 pairs of primers were designed according to the genomic sequence of L.intracellularis PHE/MN1-00 strain published in GenBank.The specificity of the primers was verified by SYBR GreenⅠfluorescent dye PCR.The corresponding TaqMan probes were synthesized according to the specific upstream and downstream primers with the best amplification efficiency,the reaction conditions were optimized,and their linear range,sensitivity and repeatability were verified.The results show that the established TaqMan fluorescence quantitative PCR method had strong specificity;when the target nucleic acid was 2.95×10^(1)-2.95×10^(6) copies/μL,the linear relationship was good,the correlation coefficient was 0.9981,and the amplification efficiency was 96.51%;the lowest detection concentration was 5 copies/μL;the coefficient of variation between and within batches was less than 2%,and the repeatability was good.373 fecal samples collected from pig farms in Jiangxi Province from October 2020 to February 2021 were tested by conventional PCR and qPCR.The total detection rate of L.intracellularis was 21.4%(80/373)and 27.6%(103/373),respectively.It showed that qPCR had higher sensitivity and the positive rate of pig farm was 88.9%(8/9).The positive rates of parturient sows,nursery piglets and fattening pigs were 28.7%,36.4% and 14.3%,respectively.The test results provide a reference for the prevalence of L.intracellularis in Jiangxi Province.

关 键 词：胞内劳森菌 TaqMan实时荧光定量PCR 检测 

分 类 号：S852.61[农业科学—基础兽医学]