Reverse transcription-multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi.
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.


CONCLUSIONS
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.
Morin
Nicole J
NJ
Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Gong
Zhilong
Z
Li
Xing-Fang
XF
D001426:Bacterial Proteins / Q000235:genetics*; D002238:Carbohydrate Epimerases / Q000235:genetics; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000235:genetics; D008070:Lipopolysaccharides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012485:Salmonella typhi / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D000637:Transaminases / Q000235:genetics; D041241:Vibrio cholerae O1 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D014871:Water Microbiology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase