利用实时荧光定量聚合酶链式反应快速检测人及动物粪便中的沙门菌

目的为了快速检测、鉴定沙门菌属细菌,提高食源性疾病暴发应对能力,本研究建立了针对沙门菌属的实时荧光定量聚合酶链式反应(qPCR)检测方法,并对其特异性、敏感性和检测下限进行评价。方法筛选针对沙门菌的属特异基因,并建立该基因的qPCR检测体系,利用肠道不同种属细菌、不同亚种及血清型沙门菌属细菌、动物及人粪便样本评价该体系的特异度、灵敏度及检测下限。结果获得沙门菌的属特异基因ttrA,建立基于该基因的qPCR检测方法。发现该方法对纯DNA的最低检测下限为2拷贝/反应。对沙门菌属以外的肠道致病菌无扩增,对1 100株不同亚种及血清型的沙门菌的扩增结果均为阳性,对150份沙门菌致腹泻患者的粪便增菌液和210份动物带菌粪便增菌液均检测阳性。结论本研究建立的基于单一基因的沙门菌属快速分子检测方法具有特异度高、灵敏度高的特点,可用于快速筛查、鉴定沙门菌及由其引起的感染性腹泻。

Establishment of a real-time fluorescence quantitative polymerase chain reaction assay for rapid detection of Salmonella in stool samples of humans and animals

ObjectiveTo rapidly detect and identify Salmonella and improve the response to the outbreaks of foodborne disease, we established a real-time fluorescence quantitative polymerase chain reaction (qPCR) assay. The specificity, sensitivity and detection limit of the assay were evaluated.MethodsFirstly, we screened some specific genes of Salmonella by using conventional PCR, then established a qPCR assay targeting one specific gene selected. The specificity, sensitivity and detection limit of the qPCR assay were evaluated by pure strains from different species of enterobacteria, Salmonella strains in different species and serotypes, and stool samples from humans and animals.ResultsOne specific gene, ttrA, was selected as the candidate gene for establishing PCR assay to detect Salmonella. The detection limit of the qPCR assay with purified DNA as template was 2 copies per reaction. The amplification results of qPCR assays were positive for all 1 100 Salmonella isolates of different species and serotypes, while the results were negative for all the strains of other enterobacteria. For 360 samples of stool enrichment, including 150 Salmonella culture positive human stool samples and 210 Salmonella culture positive animal stool samples, the qPCR detection results were all positive.ConclusionA qPCR assay based on a single gene to identify and detect Salmonella was established for the molecular detection and characterized by high specificity and high sensitivity. It can be used for the rapid detection and identification of Salmonella and the diagnosis of infectious diarrhea caused by Salmonella.