德尔卑沙门氏菌血清型分子检测靶点筛选及多重PCR检测方法的建立

Screening serotype specific genes and developing a multiple PCR method to determine Salmonella enterica serovar Derby

利用比较基因组技术筛选到德尔卑沙门氏菌(Salmonella Derby, SD)的血清型特异性基因,以此为靶点设计引物与139-141引物共同构建多重PCR检测体系,对其特异性、菌落灵敏度、抗干扰能力及人工污染等方面进行评价。当扩增出171、284和512 bp电泳条带时,检测结果为阳性。通过该方法检测39株沙门氏菌和18株非沙门氏菌,表现出100%特异性,该检测体系的DNA灵敏度为383.2 pg/μL,菌落灵敏度为52 CFU/mL。当SD与自然(猪肉、鸡肉和牛肉)的背景菌群浓度比为1∶10~4时,该检测体系能获得清晰、准确的3条扩增条带。在人工污染的猪肉、鸡肉和牛肉样品的检测中,增菌10 h,检测灵敏度为3.8 CFU/25g。该检测方法准确灵敏的检测SD,可在食品安全领域得到广泛应用。

In this study, serotype-specific genes of Salmonella enterica serovar Derby(SD)(RU6100441, RU6100445, RU6100447, RU61RS09205, and RU61RS06985) were identified by bioinformatics and comparative genomics analysis. Primers based on RU6100441 and RU6100447, as well as 139-141 primer were used to develop a multiplex polymerase chain reaction(PCR) assay. The assay was evaluated by its specificity, colony forming sensitivity, anti-interference ability, and artificial contamination. Positive results were showed with three specific bands: 171 bp, 284 bp, and 512 bp. There were 39 Salmonella strains and 18 non-Salmonella strains detected by this method, showing 100% specificity. The detection limits of this assay for DNA and colony forming were 383.2 pg/μL and 52 CFU/mL, respectively. When the background colony concentration ratio of SD to nature(pork, chicken, and beef) was 1∶104, three clear and accurate amplification bands were obtained. In artificially contaminated pork, chicken, and beef samples, this assay could detect as few as 3.8 CFU/25 g after 10 h enrichment. This method can detect SD accurately and sensitively, and therefore can be widely used for food safety.