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Page No. : 93-99

ABSTRACT

Efficient sample treatment methods are needed to fully exploit the potential of the PCR technique to detect Salmonella in food. Six different template preparation methods viz., genomic DNA extraction, heat lysis, sonication and lysis buffers -1, 2 and 3 namely, SDS-Triton X-100, TritonX-100 and NAOH SDS, respectively, were compared to determine the most sensitive, rapid and simple method, efficient in recovering Salmonella DNA from broth culture for use in PCR assay. It was observed that template prepared by isolation of genomic DNA gave a clear-cut amplicon of 389 bp length. Amplicons of equivalent intensity were also obtained with sonication and heat lysis (boiling and snap chilling) methods. Template prepared by lysis buffer 1 and 2 also gave a positive amplicon but of lesser intensity. However, template preparation using lysis buffer 3 did not yield any PCR product. Therefore, it was concluded that boiling followed by snap chilling is simple, rapid and most efficient DNA template preparation method for PCR based detection of Salmonella. In addition, six different enrichment broths were evaluated to assess their PCR compatibility. Two non-selective broths (brain heart infusion broth (BHI) and buffered peptone water (BPW)) and four selective enrichment broths (Rappaport Vassiliadis (RV), Muller Kauffmann tetrathionate (TT), selenite cystine (SC) and Salmonella enrichment broth (SEB) were inoculated with S. Typhimurium culture, incubated (37C, 18 h) and subjected to PCR after heat lysis (treatment-1). The PCR assay gave positive result with the two non-selective broths (BPW and BHI) and one selective broth (SEB), whereas, the other three broths gave negative results. Later the four selective broth cultures (RV, TT, SC and SEB) were subjected to four different treatments and PCR performed. It was observed that RV, TT and SEB gave positive result in all the treatments, whereas SC gave result in PCR assay, only after treatment 4.

Keywords: Enrichment broths, Salmonella, PCR, template preparations.