Development of an ultra rapid and simple multiplex polymerase chain reaction technique for detection of Salmonella typhi

OBJECTIVE: To make a rapid and definite diagnosis of Salmonella enteritis, using an ultra rapid multiplex polymerase chain reaction (PCR) detection method for major Salmonella serotypes, such as Salmonella typhi, Salmonella Typhimurium and Salmonella Havana.

METHODS: We performed this study at the Research Center of Molecular Biology, Institute of Military Medicine, Bagyatallah University of Medical Sciences, Iran from June 2004 to July 2005. The PCR primers for tyv (rfbE), prt (rfbS) and invA genes were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. By using simple DNA extraction method in 10 minutes, rapid PCR cycles with total cycle times of 35 minutes and rapid electrophoresis procedure with simple and very cheap buffer used in 200 to 300 volts for 15 minutes to separate the PCR products.

RESULTS: The results showed that all reference and clinical isolates of Salmonella serovars Typhi and Paratyphi were accurately identified by this assay. Specificity analysis revealed no cross-reaction with other Enterobacterial strains. The sensitivity of the PCR and the multiplex PCR was 1-10 cells. The total time of Multiplex PCR from sample preparation to final result is 45-50 minutes.

CONCLUSION: These data indicate that the specificity and sensitivity of the PCR and the multiplex PCR make them potentially valuable tools for diagnosis of Salmonella typhi bacteria and that they may be used for the identification of Salmonella enteritidis responsible for sporadic enteritis cases.