Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR

Abstract

BackgroundThermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR.ResultsSpecies specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low.ConclusionsProposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.

Rapid Molecular Approach for Simultaneous Detection of Salmonella spp., Shigella spp., and Vibrio cholera

“…In many research studies, multiplex PCR has been applied for rapid identification of diarrheal agents 25, 26. All of these studies noted that multiplex PCR is a reliable, useful, and cost-effective assay, which is consistent with our results.…”

Cooked poultry meat and products as a potential source of some food poisoning bacteria

“…There are numerous transmission routes for Salmonellosis, but the majority of the human infections are derived from consumption of contaminated poultry products (Saha et al, 2016). Thermo-tolerant bacteria belonging to Campylobacter genus (especially C. jejuni, C. coli, C. lari and marginally C. upsaliensis) are recognized as leading human foodborne pathogens causing an acute gastrointestinal disease called campylobacteriosis ) Vondrakova et al, 2014).Chicken meat must be of a high microbiological quality in order to ensure that the consumer receives a product that is not spoilt, or does not carry foodborne disease.…”

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

“…Campylobacter only (Wiemer et al, 2011;Buchan et al, 2013;Vondrakova et al, 2014). This is an advantage of this study as the listed Campylobacter species have been shown to cause a number of infections (EFSA, 2015a,b).…”

“…The qPCR assays which included the Campylobacter 16S rRNA gene rrna among targets, enabled to detect three Campylobacter species, namely C. jejuni , C. coli and C. lari in the multiplex conditions along with other foodborne pathogens in contrast to previously published multiplex detection systems developed for Campylobacter only (Wiemer et al ., ; Buchan et al ., ; Vondrakova et al ., ). This is an advantage of this study as the listed Campylobacter species have been shown to cause a number of infections (EFSA, ,b).…”