A molecular beacon-based duplex real-time polymerase chain reaction assay for simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes in milk and milk products.

In this study, a real-time polymerase chain reaction assay based on two specific molecular beacons tagged with different reporter dyes was designed and developed for Escherichia coli O157:H7 and Listeria monocytogenes in such a way that each pathogen could be detected simultaneously in a single tube and differentiated. The duplex assay was developed by targeting the rfb gene of E. coli O157:H7 and the hly gene of L. monocytogenes using the homemade master reaction mix. The detection limit of the assay in reconstituted nonfat dried milk (11%) spiked with the two targeted pathogens at different levels was 1 and 3 log colony forming units/mL of each with and without enrichment (6 h) of the sample. The assay was quantifiable for both pathogens over 5 logs with respective regression coefficient 0.9852 (E. coli O157:H7) and 0.9812 (L. monocytogenes). The application of the developed assay on 60 market samples, including 20 samples of two popular Indian indigenous products (10 each of Kulfi and Paneer), revealed three samples involving one each of raw milk, kulfi, and paneer found to be positive for E. coli O157:H7, while one sample of raw milk was positive for L. monocytogenes. The performance of the assay was validated using commercially available individual detection kits for both pathogens, which further authenticated the results by detecting the same samples positive. These assays were set up rigorously in a closed system, therefore enabling rapid, highly specific, and sensitive detection of E. coli O157:H7 and L. monocytogenes in dairy food samples.