改良分子信标-实时荧光PCR同时检测猪霍乱沙门菌和丙型副伤寒沙门菌  

Development of modified molecular beacon based real-time PCR assay for the rapid detection of Salmonella choleraesuis and Salmonella paratyphi C

作　　者：林一曼 石晓路 朱玉梅[2] 邱亚群 李迎慧 吕东月 扈庆华 

机构地区：[1]深圳市疾病预防和控制中心,深圳518055 [2]深圳市慢性病防治中心

出　　处：《卫生研究》2011年第4期514-516,共3页Journal of Hygiene Research

基　　金：国家科技重大专项(No.2008ZX10004-006)

摘　　要：目的建立猪霍乱沙门菌和丙型副伤寒沙门菌的实时荧光PCR快速检测方法,应用于食品和食物中毒病人标本的猪霍乱沙门菌检测以及沙门菌属C群的鉴别诊断。方法根据GenBank公布的猪霍乱沙门菌和丙型副伤寒沙门菌的保守序列(CP000857.1),设计引物和改良分子信标探针,优化PCR程序和PCR成分,以11种细菌为对照,建立实时荧光PCR检测方法,并用71株常见血清型的沙门菌评价该方法的灵敏度和特异性,所建立的方法应用于70份食品样品的检测。结果该改良分子信标—荧光PCR方法的DNA最低检出限为10fg/反应体系,菌液最低检出限为20 CFU/反应体系;无交叉荧光信号产生。针对目标菌均出现特异荧光信号。对71株沙门菌属的检测,该方法的灵敏度和特异度为100%。70份食品样品用荧光PCR和传统方法检测猪霍乱沙门菌和丙型副伤寒沙门菌,结果均为阴性。该方法从菌株的核酸提取到检测仅需1.5小时,从样品处理到检测仅需1天时间。结论建立的实时PCR检测方法快速、灵敏度高、特异性强,可用于食品及食物中毒标本的猪霍乱沙门菌检测以及沙门菌属C群的鉴别诊断,保障食品安全。Objective To develop real-time PCR assay based on modified molecular beacon for simultaneous detection of S.choleraesuis and S.paratyphi C.The established method was applied to the rapid detection of S.choleraesuis in food and stool samples of food poisoning,and then was applied to the identification of Salmonella C.Methods Based on the sequences（CP000857.1）published in GenBank,Two sets of primers and modified molecular beacon were designed.The Real-time PCR assay for the simultaneous detection of S.paratyphi C and S.choleraesuis was developed with optimized PCR procedures and PCR components,while other 11 different bacterial species were as the control.Then the sensitivity and specificity of the assay were tested using 77 Samonella strains.The assay was applied to the detection of 70 food samples.Results The limit of detection achieved was 10fg/reaction or 20 CUF/reaction,Only Salmonella paratyphi C and Salmonella choleraesuis strains generated fluorescent signals.No cross-reaction was observed with other 11 bacterium.the sensitivity and specificity were both 100%.No samples among 70 food samples were found Salmonella positive by both real-time PCR assay and traditional culture method.It could be finished within 2 hours from template preparation to detection.and the overall test would be finished within one day.Conclusion The real-time PCR assay was rapid,sensitive and specific.It could be applied to the rapid diagnosis of S.paratyphi C and S.choleraesuis in food and stool samples of food poisoning and the identification of Salmonella C to guarantee food safety.

关 键 词：改良分子信标 实时荧光PCR 猪霍乱沙门菌 丙型副伤寒沙门菌 

分 类 号：R155.51[医药卫生—营养与食品卫生学]