利用实时荧光定量聚合酶链式反应快速检测鸭沙门菌

Application of real-time fluorescence quantitative polymerase chain reaction for rapid identification and detection of Salmonella Anatum

目的为快速检测、鉴定鸭沙门菌,避免血清型误判,提高鸭沙门菌暴发应对能力,建立一种实时荧光定量聚合酶链式反应(qPCR)检测方法。方法随机挑选60株鸭沙门菌及238株肠道常见病原菌代表菌株,针对鸭沙门菌血清型特异基因AW58_15605设计引物并建立qPCR检测体系,通过纯菌菌株、模拟粪便样本及临床样本评价该体系的特异度、灵敏度及检测下限。结果 60株鸭沙门菌株及临床感染的50份样本扩增结果均为阳性,而对鸭沙门菌血清型以外的其他沙门菌及肠道菌的扩增均为阴性。对鸭沙门菌纯菌DNA的最低检测下限为8拷贝/反应。粪便模拟样本检测中,增菌后qPCR检测下限达59.10cfu/g,明显低于分离培养及增菌前。结论本研究建立的基于特异基因的鸭沙门菌分子检测方法具有可操作性强、特异度高、灵敏度高的特点,建议在应急情况下优先选择qPCR用于沙门菌暴发筛查、鉴别及诊断由其引起的腹泻性疾病。

Objective To establish a quantitative real time PCR(qPCR)for rapid identification and detection of Salmonella Anatum to prevent bacterial serotype misdiagnosis and improve outbreak response.Methods A total of 60 strains of S.Anatum and 238 reference strains of common enteric pathogens were selected randomly.Primer sets targeting the specific gene AW5815605 of S.Anatum were designed and a qPCR assay based on DNA molecules was established.The specificity,sensitivity and detection limit of the qPCR assay were evaluated by using pure strains,simulated stool samples and clinical stool samples from diarrheal patients.Results The qPCR amplification results indicated that the 60 S.Anatum strains and 50 clinical S.Anatum culture positive stool samples were all positive for S.Anatum specific gene AW5815605,but negative for the specific gens of other Salmonella serotypes and enteric pathogens.The detection limit for purified S.Anatum DNA was 8 copies/reaction.After enrichment the detection limit of the qPCR assay significantly decreased to 59.10 cfu/g with extracted DNA as template for the simulated human stool samples,which was significantly lower than those of pre-enrichment and isolation method.Conclusion In this study,a qPCR assay based on a specific gene AW5815605 was established to identify S.Anatum with high specificity and high sensitivity,and the assay can be used for rapid screening and identification of S.Anatum and diagnosis of related diarrheal diseases.