A scorpion probe-based real-time PCR assay for detection of E. coli O157:H7 in dairy products

Abstract

Methods for rapidly detecting Escherichia coli O157:H7, a high risk foodborne pathogen of considerable public health concern, are extremely important for monitoring of food safety. Conventional detection such as microbiological, biochemical, and immunological methods, including polymerase chain reactions (PCR), are time consuming and only qualitative. In this study, a rapid real-time PCR (RTi-PCR) method based on Scorpion probe technology targeting the eae gene of E. coli O157:H7 was developed and applied using spiked and naturally contaminated products. The assay was highly specific and did not show any cross-reactivity with any of the 15 nontargeted bacterial cultures. The assay was sensitive enough to detect 2 log colony-forming units (CFU)/mL of the target pathogen in pure broth culture and 3 log CFU/mL in spiked skim milk. When the assay was applied with 60 market dairy food samples, one sample each of raw milk, paneer, and ice cream, was found to be positive for E. coli O157:H7 with respective RTi-PCR counts of 6.7, 6.2, and 5.9 log CFU. The present study demonstrates the application of Scorpion probe technology for the detection of foodborne pathogens and reports the presence of E. coli O157:H7 in paneer, a very popular indigenous dairy product (soft cheese) in India, for the first time.