Detection and identification of Escherichia coli O157:H7 by multiplex real-time PCR
10.3760/cma.j.issn.0254-5101.2009.12.023
- VernacularTitle:多重实时荧光定量PCR检测肠出血性大肠杆菌O157:H7
- Author:
Dazhi JIN
;
Zheng ZHANG
;
Yun LUO
;
Suyun CHENG
;
Min ZHU
;
Julian YE
- Publication Type:Journal Article
- Keywords:
Multiplex real-time PCR;
Escherichia coli O157: H7;
Detection
- From:
Chinese Journal of Microbiology and Immunology
2009;29(12):1135-1139
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a rapid, sensitive and specific assay based on multiplex real-time PCR for detecting and identifying Escherichia coli O157: H7. Methods The lipopolysaccharide gene (rJbE) and H7 flagellar antigen gene(fliC) of Escherichia coli O157:H7 was chosen as targets, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and HEX fluo-resceins respectively; the 3'end of probes was labeled with MGB. The PCR reaction was optimized systemati-cally. Then the specificity, sensitivity and reproducibility of multiplex real-time PCR were estimated. Final-ly, multiplex real-time PCR was applied to detected clinical specimens. Results Escherichia coil O157:H7 were detected by multiplex real-time PCR accurately and quickly, which could distinguish Escherichia coli O157:H7 from O157: non-H7. Meanwhile, none of other bacteria could be identified. The sensitivity was 10 CFU/ml in pure culture. The coefficient of variation of intra-assay and inter-assay was less than 5%. When this assay was applied directly to identify 66 clinical specimens, the results showed that t5 were positive to Escherichia coil O157:H7 and 2 were positive to Escherichia coil O157: non-H7, in which 16 was the same to the results obtained from the conventional assays. The coincidence was 98.49%. Conclusion It is showed that multiplex real-time PCR is a reliable, accurate and feasible assay for detecting and identifying Escherich-ia coli Oi57: H7, The assay reported here provided a tool for analysis and diagnosis in the field of detecting clinical pathogens, epidemiologic survey and food safety monitoring.
