沙门菌毒力基因多重PCR检测方法的建立及应用

Development and application of multiplex PCR for detection of virulence genes in Salmonella

通过建立沙门菌不同毒力岛基因的多重PCR,对沙门菌毒力基因进行快速、灵敏的检测。根据GenBank中的序列设计合成16对引物,优化反应条件,建立了4组多重PCR,通过倍比稀释模板检测多重PCR的敏感性。利用建立的多重PCR对124株沙门菌进行毒力基因分布检测,验证多重PCR的实用性。电泳结果显示,多重PCR体系可有效扩增出每个目的基因。4组多重PCR体系的DNA敏感性分别为50、10、50、100pg;菌液敏感性分别为1×104、1×104、1×105、1×105 CFU。多重PCR与单基因PCR检测沙门菌毒力基因的结果一致。结果表明,本研究建立的多重PCR可有效检测沙门菌毒力岛基因,特异性强,灵敏度高,耗时短;还可用于沙门菌毒力基因的鉴定及分子流行病学调查。

To detect virulence genes in Salmonellapathogenicity island,a rapid and sensitive multiplex PCR was developed.A total of 16 primer pairs were designed according to sequences from the GenBank,4multiplex PCR assays had been established by optimizing reaction conditions.The sensitivity and specificity of the multiplex PCR were also determined.The practicability of virulence genes were tested in 124 Salmonellastrains using the multiplex PCR assay.Electrophoresis showed that each gene was effectively by the multiplex PCR.The DNA sensitivity of 4multiplex PCR assays was 50,10,50 and 100pg,and the bacteria sensitiviy was 1×104,1×104,1×105 and 1×105 CFU,respectively.In conclusion,the developed multiplex PCR assays are more specific,sensitive and rapid for the detection of Salmonella virulence genes,and are able to be used for bacterial identification and epidemiological investigation.