猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用  

Development of A Rapid Duplex Real-Time PCR Assay for Streptococcus suis and S.suis Serotype 2

作　　者：吴静波[1] 南文金[1] 黄健强[1] 胡鸿惠[1] 彭国良[1] 彭凌[1] 董小英[1] 

机构地区：[1]韶关学院粤北生猪生产及疫病防控协同创新发展中心,韶关512005

出　　处：《畜牧兽医学报》2018年第2期368-377,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省粤北生猪生产与疫病防控协同创新发展中心项目;广东省现代农业产业技术体系建设专项(生猪创新团队);韶关市科技计划项目(韶科[2016]44号);韶关学院科研项目(314-140694);广东省科技计划项目(2017A020225044)

摘　　要：为快速区分检测猪链球菌血清型2型和其他血清型,以猪链球菌的保守基因gdh和2型特异性基因cps2J为靶基因设计引物和TaqMan探针,建立猪链球菌通用型和2型特异性的双重荧光定量PCR检测方法,并与常规PCR方法一起对临床样品进行检测。结果显示:本方法在1.5h内可完成猪链球菌和2型猪链球菌同时检测,与其他细菌无交叉反应;检测灵敏度可达5拷贝数,标准曲线相关系数大于0.999,批内和批间CV均小于1.25%。对67份临床样品检测显示猪链球菌和2型猪链球菌检出率分别为79.1%和35.8%,与常规PCR检测结果的符合率为92.5%和89.6%,kappa值为0.800和0.757,具有极好的一致性。成功建立了灵敏、特异和稳定的双重荧光定量PCR方法,实现了猪链球菌和2型猪链球菌同时及快速诊断。In order to detect S.suis and S.suis serotype 2 rapidly,two sets of specific primers and TaqMan probes were designed according to the gdhand cps2 Jgene of S.suis serotype 2 to establish a duplex real-time PCR assay for detection of S.suis and S.suis serotype 2.Then the duplex real-time PCR was used to detect clinical samples and compare with conventional PCR.Results showed that S.suis and S.suis serotype 2 could be identified in 1.5 hours by the duplex real-time PCR assay and without any cross reaction with other bacteria.Besides,the detection limit was 5 copies/reaction.The correlation coefficient of standard curves was over 0.999,and intra-and inter-assay CV was less than 1.25%.Positive rate for sixty-seven clinical samples was 79.1% and35.8%,respectively.Coincidence rates of duplex real-time PCR with conventional PCR for S.suis and S.suis serotype 2 were 92.5%and 89.6%,kappa values were 0.800 and 0.757,respectively.A sensitive,specific,reproducible assay is established successfully to detect S.suis and S.suis serotype 2 simultaneously and rapidly,and this assay is very suitable for detection of mass clinical samples.

关 键 词：猪链球菌 2型猪链球菌 双重荧光定量PCR 快速检测 

分 类 号：S852.611[农业科学—基础兽医学]