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Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

¹ Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

^aAddress correspondence to this author at: Department of Public Health Sciences, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta T6G 2G3, Canada. Fax 780-492-7800; e-mail xingfang.li{at}ualberta.ca.

Background: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens.

Methods: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates.

Results: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis.

Conclusion: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

The following articles in journals at HighWire Press have cited this article:

				Y. Liu, A. Gilchrist, J. Zhang, and X.-F. Li Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water Appl. Envir. Microbiol., March 1, 2008; 74(5): 1502 - 1507. [Abstract] [Full Text] [PDF]

				A. J. Gubala and D. F. Proll Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae Appl. Envir. Microbiol., September 1, 2006; 72(9): 6424 - 6428. [Abstract] [Full Text] [PDF]