Abstract
Introduction and objective: Salmonellosis is responsible for large numbers of infections in both humans and animals. Conventional methods of isolation of Salmonella strains take 4-7 days to complete and are therefore laborious and require substantial manpower. Our main objective was to develop a rapid detection method using shortened PCR cycles in a conventional thermal cyclers and fast electrophoresis for Salmonella.
Materials and methods: The PCR primers for tyv (rfbE), prt (rfbS) and invA, genes were used for the rapid identification of S. enterica serovars typhi and paratyphi A, with rapid and short cycles of multiplex PCR. By using very fast and simple DNA extraction method in 10mins, rapid PCR cycles with total times of 35mins and rapid electrophoresis procedure with simple and very cheap buffer in 15mins we were able to separate the PCR products.
Results: All references and clinical isolates of Salmonella serovars typhi and paratyphi were accurately identified. Specificity analysis revealed no cross reaction with other enterobacterial strains. The sensitivity of the assay was 1-10 cells. The total time of multiplex PCR from sample preparation to final result is 45 to 50mins.
Conclusion: These data indicate that the specificity and sensitivity of optimized rapid cycle multiplex PCR is a potentially valuable tool for rapid diagnosis of S. typhi using a conventional thermal cycler. This method cut the time of a PCR reaction from 3.5h to less than 60mins. These findings could also be applied to other PCR programs detecting various genes allowing researchers to significantly shorten their PCR reaction times.
Keywords
Rapid cycle Multiplex PCR Salmonella typhi Conventional thermal cyclers Rapid electrophoresis
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