Rapid Detection Of viable Salmonella On Poultry Carcasses Without Enrichment Using Multiplex PCR And Quantitative PCR (qPCR)

By Steven C. Ricke

Foodborne salmonellosis is a public health concern and poultry and poultry products are often regarded as a primary source of infection. Salmonella species can be introduced and distributed on carcasses during processing and even low numbers of Salmonella on products can represent a health risk in humans.

The overall objective of this project was to detect and quantify viable Salmonella serovars in poultry carcasses without selective enrichment using multiplex PCR and quantitative PCR (qPCR). The specific objectives were a) to optimize both multiplex PCR and qPCR assays for simultaneous detection of Salmonella genus, Salmonella subspecies I, S. enteritidis, S. Heidelberg and S. typhimurium; b) apply the PCR assays to poultry carcass rinsates for identification of viable Salmonella without enrichment.

It was found that the growth of different Salmonella serovars was most similar and detectable levels occurred much sooner on nonselective Luria Bertani broth than selective enrichment with either RV broth or tetrathionate broth. For PCR assays, using nonselective media that supports the most rapid growth of Salmonella can greatly shorten assay time.

There is indication of competition between serovars during growth. In particular, S. typhimurium exhibited decreased growth in S. Heidelberg (ARI-14) spent media. This could impact current enrichment and most probable number (MPN) enumeration assays and suggests that incubation periods in nonselective media should not be allowed to be extensive as underestimation of certain serovars could occur.

In the development of the PCR assays S. Heidelberg specific primers that could detect 15 strains isolated from poultry, poultry products and humans were determined. The multiplex PCR assay using five primer pairs for Salmonella genus (423 bp), Salmonella subspecies I (137 bp), S. enteritidis (171 bp), S. Heidelberg (216 bp) and S. typhimurium (310 bp) was standardized.

The quantitative PCR was optimized to detect as low as 46 copies of Salmonella genomic DNA. It was confirmed that the two PCR assays, when applied to the detection of Salmonella on contaminated chicken breast meat, detected 22 CFU per gram after eight hours of nonselective incubation.