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Page No. : 113–124

ABSTRACT

A set of primers (Salm-3 and Salm-4) targeting genus-conserved invA was employed to standardize PCR assay for detection of Salmonella from foods, which gave specific amplification of a 389 bp fragment. On restriction endonuclease analysis with HinfI enzyme, S. Typhimurium gave restriction fragments of 242 bp and 147 bp whereas other serotypes gave fragments of different lengths. The standardized PCR protocol was found to be specific when tested with 61 strains of Salmonella, belonging to 48 different serotypes which gave a specific 389 bp product, whereas 13 other non-Salmonella bacteria did not yield any product.
The minimum detection level with pure bacterial culture was found to be 3.7 cfu per ml. Experimental inoculation studies carried out over varied incubation schedule employing buffered peptone water as pre-enrichment broth followed by selective enrichment in four different selective broths revealed that earliest detection time by PCR of Salmonella from spiked meat was 20 h. Further, salmonella enrichment broth and tetrathionate broth were the most suited selective broths for the assay. There was good agreement between the results of PCR and cultural isolation. Examination of 75 naturally contaminated meat samples (49 beef and 26 chicken) revealed one chicken sample to be positive by PCR. The standardized two-step enrichment PCR protocol holds promise as a reliable and rapid method for the detection of Salmonella from foods.

Keywords: Salmonella, PCR, sensitivity, specificity, spiking