Development and application of a new multiplex real-time PCR assay for simultaneous identification of Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi-PCR for the detection of B.melitensis, C.sakazakii and L.monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n=25) and cheese samples (n=20) were analysed by multiplex RTi-PCR assay and detected for quantification of the three pathogens. In this study, B.melitensis, C.sakazakii and L.monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi-PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r(2)=0.986-0.997). Multiplex RTi-PCR results showed that most of the samples were contaminated with the pathogens screened.