Rapid detection and specific differentiation of Salmonella enterica subsp. enterica Enteritidis, Typhimurium and its monophasic variant 4,[5],12:i:− by real-time multiplex PCR

•A TaqMan 5-plex real-time PCR method was developed and validated.•It detects specifically S. Enteritidis, S. Typhimurium and its monophasic variants.•The inclusivity and exclusivity were between 98 and 99%.•The limit of detection was 3–5 CFU/25 g neck skin sample.•The method is useful for detecting serovars mentioned in regulation (EU) no 1086/2011.

Salmonella enterica is one of the most common zoonotic pathogens worldwide causing clinical diseases in human and animal hosts. Targeting a reduction of Salmonella prevalence in poultry, the EU set up a microbiological criterion that demands the absence of S. enterica subsp. enterica serovars Enteritidis and Typhimurium including its monophasic variant with seroformula 4,[5],12:i:− in 25 g of poultry neck skin samples and fresh meat according to regulation (EU) no 1086/2011. We developed and in-house validated a method that detects and differentiates these Salmonella serovars based on a 5-plex real-time PCR assay within 24 h after sampling. The inclusivity and exclusivity were between 98 and 99% analysing 456 bacterial strains. Validation according to ISO 16140:2003 against the traditional cultural reference method ISO 6579:2002 was performed using 60 artificially contaminated and 31 presumably naturally contaminated chicken neck skin samples resulting in a relative accuracy of 100%. The detection probability reached 100% between 3 and 5 CFU/25 g sample. We were also able to assign rough and non-motile strains to S. enterica subsp. enterica serovars Enteritidis and Typhimurium. In conclusion, we provide diagnostic laboratories a fast and accurate method to monitor these Salmonella serovars in chicken neck skin samples. Other matrices could be easily adapted.