A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157:H7

作者：Yang, X.; Cheng, H. W.; Chen, L.; Zhao, J.; Chang, H. T.; Wang, X. W.; Liu, H. Y.; Yao, H. X.; Zhang, L. X.; Wang, C. Q.^*

来源：Genetics and Molecular Research, 2013, 12(4): 4836-4845.

DOI：10.4238/2013.October.22.3

• Duplex
• SYBR Green
• Quantitative PCR
• Escherichia coli O157:H7

PCR and hybridization assays are widely used for the detection and identification of Escherichia coli serogroups and serotypes. We used these techniques for the detection of E. coli O157:H7, a dominant serogroup among E. coli strains that are considered major public health problems worldwide. We developed a quantitative PCR assay using SYBR Green I, based on the published sequences of the rfbE and fliC genes from E. coli O157:H7. This method detected the E. coli O157:H7 O somatic antigen gene and the flagellar antigen gene simultaneously, with good specificity, sensitivity, and repeatability. The sensitivity of the assay was 2.95 x 10 copies/mu L, which is 10(3) times more sensitive than obtained with a conventional PCR. The intra-assay and inter-assay coefficients of variation were less than 2%. We concluded that this duplex quantitative PCR assay is adequate for the identification and quantitative analysis of E. coli O157:H7. This provides a new identification method for clinical diagnosis of E. coli O157:H7 and for food safety analysis, as well as for molecular epidemiological studies of foodborne diseases.

• 出版日期2013
• 单位河南农业大学