肉类中大肠杆菌O157:H7多重PCR检测方法的建立

Specific and Sensitive Detection of Escherichia coli O157:H7 in Meat by a Multiplex PCR

摘要:

以编码大肠杆菌O157抗原的rfbE基因、 编码H7抗原的fliC基因以及编码毒力因子的eaeA基因为靶基因, 选择3对引物, 建立并优化了检测大肠杆菌O157:H7的多重PCR体系, 扩增产物分别为291 bp、625 bp、368 bp, 采用30株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到91.35 pg; 在存在干扰菌鼠伤寒沙门氏菌(Salmonella?typhimurium)的情况下, 当起始污染量为1.4 CFU/mL时, 37 ℃培养6 h 即可检出。在30份肉类样品中, 有3份检出了大肠杆菌O157:H7。本研究建立的多重PCR方法可特异、灵敏地实现对大肠杆菌O157:H7的检测。

Abstract:

A multiplex PCR assay for detection of Escherichia coli O157:H7 was developed by using 3 sets of primers that specifically amplify segments of the rfbE、fliC and eaeA genes. The target genes fragment of the PCR assay were 291 bp, 625 bp and 368 bp, respectively. Analysis of 30 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 91.35 pg with genomic DNA. This multiplex PCR assay did not cross-react with the background Salmonella?typhimurium and could detect 1.4 CFU/mL in artificial inoculated meat sample after enrichment at 37℃ for 6 h. Three of 30 meat samples were detected positive. These results indicated that the multiplex PCR assay can be used for specific and sensitive detection of E. coli O157:H7.