多重荧光PCR技术快速鉴定常见15种沙门菌血清型  

Rapid identification of 15 common S.enterica Salmonella serovars by multiplex real-time PCR

作　　者：左乐 姜伊祥 万敏 江敏[1] 石晓路[1] 李迎慧[1] 邱亚群[1] 林一曼[1] 扈庆华[1] Zuo Le;Jiang Yixiang;Wan Min;Jiang Min;Shi Xiaolu;Li Yinghui;Qiu Yaqun;Lin Yiman;Hu Qinghua(Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China)

机构地区：[1]深圳市疾病预防控制中心,深圳518055

出　　处：《卫生研究》2020年第5期823-828,858,共7页Journal of Hygiene Research

基　　金：深圳医疗卫生三名工程(No.SZSM201811071);深圳市卫生计生系统科研项目(No.201602004);深圳市科技计划(No.JCYJ20160428142519080)。

摘　　要：目的建立快速高效的多重荧光PCR方法,用于鉴定常见15种沙门菌血清型。方法通过沙门菌基因组比对,发现12个膜蛋白STM4497基因可有效区分常见15种沙门菌血清型,将这12个基因分别标识为A^L基因,并设计引物探针用于建立常见15种沙门菌血清型分子鉴定方法,评估其最低检出限、特异度、可重复性等指标。并以传统血清学方法作对照,采用多重荧光PCR法对206株沙门菌进行鉴定,评估新建方法的一致性。结果多重荧光PCR法最低检出限1.1×10^-3~1.2×10^-3 ng/μL,特异度100%,相对标准偏差均<2.97%。与传统血清学方法比较,新建方法的Kappa值为0.92~1.00。结论建立的基于多重荧光PCR鉴定常见15种沙门菌血清型的方法快速、准确、特异性强。OBJECTIVE Multiplex real-time PCR for the identification of 15 Salmonella serovars was developed.METHODS Through the Salmonella genome comparison,12 membrane proteins STM4497 gene can be used to identify 15 Salmonella serovars,and these 12 genes were respectively listed as A-L genes.Then primers were designed according to A-L gene conserved sequences,and then multiplex real-time PCR was established assessed with the evaluation of the limit detection,sensitivity,specificity,and repeatability.The 206 Salmonella strains were identified using multiplex real-time PCR with the comparison of the serum slide agglutination assay.RESULTS The limit detection of multiplex PCR ranged from 1.1×10^-3-1.2×10^-3 ng/μL.The target genes were 100%specificity,and the relative standard deviation was lower than 2.97%.Compared with the serum slide agglutination assay,Kappa ranged 0.92-1.00.CONCLUSION The multiplex real-time PCR can be used to identify 15 Salmonella serovars,which is rapid,accurate and specific.

关 键 词：沙门菌血清型 多重荧光PCR 

分 类 号：R378.22[医药卫生—病原生物学] R446.11[医药卫生—基础医学]