Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers se lected from conserved sequences within a 2.3 kb randomly cloned DNA fragmen t from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belongi ng to subspecies I, II, IIIb and IT:. One strain of Salm. arizona (ssp. III a) tested negative. No PCR products were obtained from any of the 31 non-Sa lmonella strains examined. The sensitivity of the assay was 2 cfu, as deter mined by analysis of proteinase It-treated boiled lysates of Salm. typhimur ium. The performance of the assay was evaluated for environmental water, se wage and food samples spiked with Salm. typhimurium. Water and sen age samp les were filtered and filters ere enriched overnight: in a non-selective me dium. Prior to PC, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 190 ml(-1) water with b ackground levels of up to 8700 heterotrophic organisms ml(-1) and 10 000 cf u of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detect ion of < 10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rap id and simple assay provides a sensitive and specific means of screening dr inking water or environmental water samples, as well as food samples, for t he presence of Salmonella spp,