A rapid multiplex PCR assay for the simultaneous detection of four major foodborne pathogens in camel milk: Development, validation and comparison of the assay with those of culture-based methods

National production of camel milk can lead to increased health and hygiene levels in communities due to its medicinal and therapeutic characteristics as well as high similarity to human milk. However, safety and microbiological quality of raw camel milk, particularly in regions where it is consumed without pasteurization that may lead to foodborne bacterial contamination, are majorly concerned. Regarding importance of the issue, it is also necessary to provide rapid and accurate diagnostic methods to minimize detection time. This study aimed to develop and validate a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous detection of four major foodborne pathogens of Staphylococcus aureus , Enterohemorrhagic Escherichia coli (EHEC), Salmonella enterica and Listeria monocytogenes in camel milk samples collected from Gonbad-Kavoos region of northern Iran and to compare results of this method with those of culture-based methods.Totally, 196 raw camel milk samples were collected from a traditional dairy shop in Gonbad Kavoos, Golestan, Iran, under sterile conditions and analyzed using traditional culture-based methods and the newly developed M-PCR. Species-specific MPCR targeting sea gene for S. aureus , inv A for Salmonella , stx -1 for EHEC and ABC transporter permease for L. monocytogenes were performed.Phenotypic and multiplex analyses revealed contamination rates for Staphylococcus aureus , EHEC, Salmonella enterica and Listeria monocytogenes as 29.6 and 19.9%, 0 and 4.2%, 14.8 and 14.8%, and 1 and 2.6%, respectively. The M-PCR assay successfully detected all four pathogens simultaneously within a short time, demonstrating high analytical sensitivity and specificity. When benchmarked against culture, M-PCR achieved sensitivity of 97% and specificity of 100%.This novel M-PCR method can be used as a rapid, accurate and reliable tool for the simultaneous identification of several pathogenic bacteria in dairy products such as camel milk with a sensitivity and specificity of respectively 97 and 100%. Furthermore, this method is more accurate and efficient than the classical culture method, enhancing public health surveillance and food safety control measures in camel-breeding communities.