Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR.

Raymond Heymans,Elisabeth G Biesta-Peters,Greetje A A Castelijn,Claudia C C Jansen,Caroliene A M Van Heerwaarden,Menno Van Der Voort,Amir Vila,Anderson De Souza Sant'Ana

doi:10.1371/journal.pone.0206316

Raymond Heymans, Elisabeth G Biesta-Peters + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0206316

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Abstract

A multiplex quantitative PCR (qPCR) was developed and evaluated for the simultaneous detection of Salmonella spp., S. enterica serovar Typhimurium and S. enterica serovar Enteritidis in various (food) matrices. Early and fast detection of these pathogens facilitates effective intervention and prevents further distribution of contaminated food products on the market. Three primer and probe sets were designed to target the invA gene, the STM4200 gene, and the SEN1392 gene to detect and differentiate Salmonella spp., S. Typhimurium, and S. Enteritidis, respectively. The multiplex qPCR targeting these three genes was optimized for efficiency and linearity. By testing 225 Salmonella isolates and 34 non-Salmonella isolates from various sources the inclusivity and exclusivity were determined. The inclusivity of the multiplex qPCR was 100% for all Salmonella isolates, including 72 S. Typhimurium isolates, and 53 S. Enteritidis isolates. The exclusivity for Salmonella spp., S. Typhimurium, and S. Enteritidis was 100%, 94.6%, and 100%, respectively. No positive results were reported for non-Salmonella isolates. The limit of detection (LOD) for the qPCR was determined for the matrices poultry, minced meat, egg, herbs/spices, powdered milk, fish, animal feed, boot-socks with chicken feces and chicken down. LOD values for qPCR and the conventional culture methods were similar, except for the matrix boot-socks and down, for which the LOD for the conventional culture methods performed better than the qPCR method. In conclusion, the multiplex qPCR assay developed allows for rapid screening of Salmonella spp., S. Typhimurium, and S. Enteritidis in various (food) matrices.

Highlights

Enteric salmonellosis imposes a major burden on public health in both underdeveloped and industrialized countries
Many cases of non-typhoidal S. enterica infections are zoonotic since the intestinal tract of various domestic and wild animals are a natural reservoir for this pathogen [4,5]
As invA is by many considered a standard for the detection of Salmonella spp. [22], a 95 bp product for the invA gene was amplified using a newly developed primer and hydrolysis probe set that was compatible with the current multiplex format

Summary

Introduction

Enteric salmonellosis imposes a major burden on public health in both underdeveloped and industrialized countries. Serious enteric illness may be caused by cross contamination of food with fecal matter of infected animals, fecal-oral transmission from infected human, or contamination from environmental or other food sources [6,7]. These diverse means of transmission emphasize the need to maintain strict hygiene regimens concerning food handling at every stage of the food production chain to prevent cross contamination and ensure food safety [8,9]. Enterica serovars Typhimurium and Enteritidis were the most frequently reported serovars in Europe and were responsible for the majority of cases of human salmonellosis in 2011 [6,10].

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