双重PCR检测肠炎沙门氏菌方法的建立

Development of a Novel Duplex-PCR Assay for Salmonella enteritidis Detection

沙门氏菌是引起人类食物中毒事件的主要病原之一,其中,肠炎沙门氏菌因其可感染家禽、污染禽蛋而受到广泛关注。本试验根据沙门氏菌属特异性基因片段fimY和肠炎沙门氏菌特异性基因片段sdfI分别设计一对引物,对25株沙门氏菌和大肠杆菌进行双重PCR扩增,结果显示22株沙门氏菌均出现与理论值大小497bp相符的属特异性条带,作为对照的3株大肠杆菌则未显示该条带,并且7株肠炎沙门氏菌均出现与理论值大小293bp相符的特异性条带。另外,敏感性试验结果显示肠炎沙门氏菌50336和鸡白痢沙门氏菌SP1的模板浓度分别为18ng和15ng时仍能清晰扩增出特异性条带。上述结果表明建立的PCR方法具有快速简便、灵敏性高、特异性强等特点,为公共卫生、食品安全、畜牧兽医及出入境安检等多部门检测和监测沙门氏菌提供了前提基础和技术保障。

Sahnonella is a major cause of global food-borne illness,and especially,Salmonella enteritidis （S.enteritidis） has received considerable publicity due to its ability to infect poultry and contaminate eggs. A duplex-PCR method was established to detect the fimY gene specific in Salmonella species and the conservative region of sdfI gene in S.enteritidis. Experimental results showed a 497 bp fragment was specifically amplified in all twenty-two strains of Salmonella species selected and a specific 293 bp fragment was amplified from all the seven S.enteritidis strains,whereas the three control strains of Escherichia coil showed none. In addition,sensitivity test results indicated that the minimum template concentration of S.enteritidis 50336 and S.pullorum SP, for amplification could reach down to 18 ng and 15 ng,respectively. All above demonstrated the reliability and sensitivity of duplex-PCR approach, and furthermore, it facilitates reliable premise and technical support in detecting and monitoring Salmonella in departments such as public health,food safety, animal husbandry, veterinary medicine, and entry-exit inspection, etc.