鼠伤寒沙门氏菌毒力岛基因 mgtC 和 sopB双重 PCR 检测的研究

摘要

Salmonella is a zoonotic important foodborne pathogen ,a sensitive dual-PCR detection method was established is beneficial to achieve the detection of pathogenic Salmonella rapidly.In this thesis,the pathogenicity island genes of pathogenic Salmonella were as the research object and two pairs of primers which named Salmonella pathogenicity island mgtC and sopB were designed and synthesized based on the sequence of the Salmonella patho-genicity island genes.The nucleic acid of Salmonella typhimurium (ATCC9150) was screened as a template,after primer specificity test,sensitivity test of DNA template and optimize the reaction conditions ,the double PCR method of rapid differential detection for pathogenic Salmonella was successfully established.Specific test results showed that the primers mgtC and sopB of double PCR only can amplify two specific fragments of the Salmonella typhimuri-um (ATCC9150) whose size is 500 bp and 1 000 bp,respectively.The sensitivity results showed that the limit of detection was 10 cfu /mL .This double PCR method established could identify the virulence factor in Salmonella, which can achieve the detection of pathogenic Salmonella rapidly in high specificity and sensitivity .%　　沙门氏菌是人畜共患的重要食源性病原菌，建立灵敏的双重 PCR 方法有益于实现对致病性沙门氏菌（Sal－monella）的快速检测。以沙门氏菌毒力岛基因为研究对象，根据 GenBank 发表的沙门氏菌毒力岛基因序列，分别设计合成了沙门氏菌毒力岛 mgtC 和 sopB 的2对引物。以鼠伤寒沙门氏菌（ATCC9150）菌株的核酸为模板，经过引物特异性试验，DNA 模板灵敏度试验，优化反应条件，成功地建立了快速鉴别以及检测鼠伤寒沙门氏菌的双重 PCR 方法。特异性试验结果表明，引物 mgtC 和 sopB 建立的双重 PCR 仅能扩增出沙门氏菌（ATCC9150）的2段特异性片段，大小分别是500，1000 bp。敏感性试验结果表明，沙门氏菌的最低检测限为10 cfu/mL。此方法能鉴定产生毒力因子的沙门氏菌，其特异性强、敏感性高，可实现对食源性致病菌的快速检测。