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Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

Moezi, P., Kargar, M., Doosti, A., Khoshneviszadeh, M.
Journal of applied microbiology 2019 v.127 no.1 pp. 262-273
Escherichia coli O157, Listeria monocytogenes, Salmonella Enteritidis, Staphylococcus aureus, cost effectiveness, detection limit, food pathogens, gene amplification, polymerase chain reaction, raw milk
AIMS: The aim of this study was to develop a multiplex touchdown PCR (multiplex TD‐PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. METHODS AND RESULTS: For this reason, a multiplex TD‐PCR assay with a pre‐enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD‐PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD‐PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD‐PCR method with the traditional culture, except in one or two samples. CONCLUSIONS: Hence, the proposed multiplex TD‐PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. SIGNIFICANCE AND IMPACT OF THE STUDY: Hence, due to its simplicity, cost‐effectiveness and being time‐saving, it seems that this method is reasonable and economical for rapid optimization of PCR reactions.