多重实时PCR快速同时检测沙门菌和志贺菌

目的 建立改良分子信标，多重实时PCR同时检测沙门菌和志贺菌的快速方法，应用于食源性致病菌的快速诊断。方法 根据GenBank公布的沙门菌侵袭性基因invA和ssaR基因，分别设计一对引物和改良分子信标探针，用同色荧光标记，用于同体系检测沙门菌。志贺菌根据ipaH基因的保守序列，设计引物和改良分子信标探针，加入沙门菌检测体系中，建立三重实时PCR一改良分子信标检测体系，应用于同时对沙门菌、志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果 改良分子信标一多重实时PCR反应体系DNA灵敏度为69～93fg/μl。菌液灵敏度为32～64CFU／ml或1～2CFu／PCR反应体系，无交叉反应。该反应体系同时检测134株沙门菌和67株志贺菌，均出现特异的荧光信号，两种细菌检测互不干扰。对细菌性食物中毒样本等共1100份同时进行沙门菌和志贺菌检测，569份沙门菌实时PCR阳性，其中551份沙门菌培养阳性；42份志贺菌实时PCR阳性，其中41份志贺菌培养阳性。从样品处理到检测结果仅需时间2h至1d。结论 改良分子信标-多重实时PCR检测体系快速、灵敏度高，特异性强，可用于沙门菌和志贺菌食物中毒的快速诊断，伤寒、痢疾等肠道传染病的初筛及预防医学门诊的健康人群体检，为食源性疾病的分子流行病学调查提供新的检测手段。

Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR

OBJECTIVE: Dual detection of Salmonella and Shigella using modified molecular beacons and real-time PCR was developed. The established method was applied to rapid diagnosis of Salmonella and Shigella' food poisoning, and for routine monitoring programs. METHODS: Two sets of primers were designed based on the core sequence of invA gene and ssaR gene published on GenBank to detect Salmonella, and ipaH gene were selected to detect Shigella. Three corresponding modified molecular beacons labeled with different fluorophors were designed. The molecular beacons and primer sets were tested against numerous strains from 55 different bacterial species. Then the two assays were combined to establish the dual real-time PCR assay, and were applied to the food poisoning diagnosis and surveillance. RESULTS: For the modified molecular beacons-based dual real-time PCR assay, the sensitivity achieved was 69-93 fg/microl, 32-64 CFU/ml or 1-2 CFU/PCR reaction. There was no cross-reaction with other bacteria served as control. The dual real-time PCR assay was used to detect 134 Salmonella strains and 67 Shigella strains but no false signals were observed. 1100 food poisoning samples were tested with 569 Salmonella and 42 were Shigella identified by real time PCR. Among the positive samples, 551 were detected Salmonella and 41 were Shigella by traditional culture method. The overall test could be finished within 2 hours to one day starting from sample preparation. CONCLUSION: The modified molecular beacons-based dual real-time PCR assay was rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Salmonella and Shigella' food poisoning.