Pentaplex Taqman Assay for the Detection of Pathogenic and Multidrug Resistant Strains of Salmonella

Introduction:  Overuse of antibiotics in the medical and animal industries is one of the causes for development of multidrug-resistant (MDR) food pathogens which are often difficult to treat. In the past few years, an increase in incidence of outbreaks caused by MDR Salmonella was documented. The ability to accurately and sensitively detect such pathogens in food is highly important.

Purpose:  The objective of this study was to develop a rapid multiplex real-time assay for detection of pathogenic and antibiotic resistant Salmonella.

Methods:  A pentaplex real-time PCR was designed targeting the virulence gene, invasin (inv), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. The sensitivity of the assay was determined via a standard curve with ten-fold serially diluted, 10 ng/µl to 1 fg/µl, genomic DNA. To avoid false negative results and to increase the reliability of the assay, an internal amplification control was added which was detected by a locked nucleic acid (LNA)-based probe.

Results:  The assay was able to detect 200 genomic equivalents (1 genomic equivalents = 5 fg) of Salmonella DNA when single targets were tested, while in a multiplex format, the sensitivity of the assay decreased to 2000 genomic equivalents.  The assay performed equally well on artificially contaminated samples of tomato, spinach, egg, ground turkey, ground chicken, chicken rinse, and ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7). These food samples contaminated with 10 CFU/g could be detected after 12 h of enrichment with 100% reproducibility. The detection limit for unenriched samples was 10⁴CFU/g.

Significance: The pentaplex real-time assay developed in this study can be applied as a sensitive and selective tool to detect antibiotic-resistant Salmonella, hence enhancing the safety of food.