Methodology article

Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples

Edel O'Regan¹ , Evonne McCabe² , Catherine Burgess² , Sheila McGuinness³ , Thomas Barry³ , Geraldine Duffy² , Paul Whyte¹ and Séamus Fanning¹

¹  Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland

²  Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland

³  Department of Microbiology, National University of Ireland Galway, Galway, Ireland

author email corresponding author email

BMC Microbiology 2008, 8:156doi:10.1186/1471-2180-8-156

Published:	21 September 2008

Abstract

Background

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.

Results

The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method.

Conclusion

Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.