福氏志贺菌血清型单重PCR鉴定方法的建立及应用  

Development of a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri

作　　者：张曙霞[1,2] 王建平[2] 金东[2] 陈道利[3] 刘凯[2] 王艺婷[2] 李振军[2] 徐建国[2] 孙强正[2] 

机构地区：[1]山西医科大学寄生虫学教研室,太原030001 [2]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室 [3]安徽省马鞍山市疾病预防控制中心

出　　处：《中华流行病学杂志》2012年第3期328-331,共4页Chinese Journal of Epidemiology

基　　金：北京市优秀博士学位论文指导教师科技项目（YB20098450101）;传染病预防控制国家重点实验室自主研究重点项目（2008SKLID106、2011SKLID203）;传染病预防控制重大专项（2008ZX10004-008、2008ZX10004-009、2009ZX10004-203）

摘　　要：目的建立基于O抗修饰基因的福氏志贺菌血清型PCR鉴定方法。方法根据福氏志贺菌O抗原合成及修饰特异基因设计引物，以常见的14种福氏志贺菌血清型（包括1a、1b、1c、2a、2b、3a、3b、4a、4b、5a、Y、X、Xv和F6）为标准菌株，建立福氏志贺菌血清型单重PCR鉴定方法；并以痢疾志贺菌、宋内志贺菌、鲍氏志贺菌和腹泻相关的其他菌属菌株验证特异性；应用该方法对106株福氏志贺菌临床分离株进行PCR血清分型。结果建立一种福氏志贺菌血清型单重PCR鉴定方法，通过8个PCR反应，能够鉴定目前已知的15种血清型中的14种（Xv除外）。检测灵敏度在10pg至1ngDNA（20gl反应体系）。对106株福氏志贺菌临床分离株的检测结果提示，PCR分型方法与玻片凝集法具有很高的一致性。结论本研究建立的福氏志贺菌血清型单重PCR鉴定方法具有特异性、灵敏性，可用于临床检测。Objective To develop a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella （S.） flexneri. Methods Eight pairs of primer for O-antigen synthesis and modification genes of S. flexneri were designed and used for developing an O-antigen modification gene-specific singleplex PCR assay to serotype 14 most common S. flexneri serotypes （ 1 a, 1 b, 1 c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Y, X, Xv and F6）. Bacterial pathogens which causing diarrheal disease were used for specificity detection. 106 S. j%xneri clinical isolates were serotyped by this method and compared with the slide agglutination method. Results An O-antigen modification, gene-specific singleplex PCR was developed. When six singleplex PCR reactions were performed, 14 of the 15 recognized S. flexneri serotypes were identified, except for serotype Xv. The detection threshold ranged from 10 pg to 1 ng DNA in a 20 p.1 reaction system. A high concordance between the singleplex PCR assay and slide agglutination were observed when 106 S. flexneri strains of various serotypes were analyzed with an exception that 1 serotype Y strain showed that it was carrying the additional defective gtr 1I genes. Conclusion This method showed advantages over the traditional slide agglutination methods, and was promising when under application in the following situations as clinical diagnosis.

关 键 词：福氏志贺菌 O抗修饰基因 聚合酶链式反应 血清型 

分 类 号：R446.5[医药卫生—诊断学]