A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

Abstract

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~102 to ~106 cfu mL−1 raw milk. The detection limit was ~102 cfu mL−1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.