Development of a PCR-based assay to detect Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella in milk

Abstract Raw milk is a potential source of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes , and Salmonella . A sensitive and specific method to detect these pathogens in milk will help to reduce potential health hazards posed by them. The present study was therefore aimed at developing a PCR assay to detect STEC, L. monocytogenes and Salmonella in milk. Initially, PCR conditions were standardized using purified DNA. A rapid boil method was tested to isolate DNA from bacterial cells in milk for subsequent use in PCR. Under standard conditions, the detection level for PCR was as low as 10 pg of purified bacterial DNA and 5 cfu of bacteria added to milk or phosphate buffered saline. Following overnight growth of bacteria in milk, the PCR technique detected as few as 10 2  cfu of bacteria. Primers used in the PCR assay were highly specific for individual organisms and did not show cross-reactivity with heterologous organisms. In conclusion, the PCR-based technique provides a sensitive and specific method for detection of STEC, L. monocytogenes and Salmonella in milk.