5种致泻大肠埃希氏菌实时荧光定量PCR快速检测技术

将5种致泻大肠埃希氏菌的毒力基因保守序列进行设计并合成了引物和探针,建立的多重实时荧光PCR4种试剂盒可以同时检测5种致泻大肠埃希氏菌。结果表明:4种试剂盒的12对毒力基因通过5种致泻大肠埃希氏菌的标准储备菌株均得到验证;并且12对毒力基因只对对应的致泻大肠埃希氏菌特异性起作用,对常见的食源性致病菌都无扩增曲线;escV、bfpB、stx1、ipaH和invE基因的检出限是10CFU/mL,aggR基因的检出限是102CFU/mL,astA和pic基因的检出限是103CFU/mL,而stx2A、ipaH和stIb基因菌液浓度<10CFU/mL时,均可观察到明显的"S"型扩增曲线,且线形较好;并且对抽查和委托检验的肉类、奶和动物腹泻物以及人工污染样品等40份样品进行检测,共检出11份阳性样本,与GB4789.6-2016标准检测结果相一致,表明建立的4种试剂盒方法具有良好的实用性。

Study on the rapid detection of five strains of diarrheagenic Escherichia coli by real-time fluorescence quantitative PCR

Primers, and probes were designed for the conserved sequences of five virulence genes involved in Escherichia coli diarrhea mentioned in the new standard of GB 4789.6-2016 national food safety standard.Four kits of multiple real-time fluorescence PCR were established to detect five diarrhea-causing E.coli simultaneously.The results showed that 12 pairs of virulence genes of the four kits were verified by the standard reserve strains of the five diarrhea-causing E.coli strains.In addition, these 12 pairs of virulence genes only play a role in the specific role of corresponding diarrhea-causing E.coli , and have no amplification curve for common foodborne pathogens.The detection limit of escV , bfpB, stx 1 , ipaH and invE genes is 10 CFU/mL, and that of aggR gene is 10 2 CFU/mL.The detection limit of astA and Pic genes was 10 3 CFU /mL.When the concentration of stx 2 A , ipaH and stIb genes was less than 10 CFU/mL, a significant "S" type amplification curve could be observed, and the line shape was better.In addition, 40 samples including meat, milk, animal diarrhea, and some artificial contamination samples were tested, and a total of 11 positive samples were found.Consistent with the test results of the new standard 4789.6-2016, The results showed that the four kit methods were simple, specific, sensitive and practical.