DNA concentration by solid phase reversible immobilization improves its yield and purity, and detection time of E. coli O157:H7 in foods by high resolution melt curve qPCR

Abstract

For molecular detection of foodborne pathogens to be effective, an efficient technique for bacterial DNA concentration is required. Solid phase reversible immobilization (SPRI) paramagnetic beads can concentrate DNA from foods containing low amounts of bacteria because of their highly specific DNA-binding capacity. This study aimed to develop an upstream bacterial DNA concentration step using SPRI beads and compare its efficacy with immunomagnetic separation (IMS) for the detection of Shiga toxin producing E. coli (STEC) O157:H7 in food samples via a multiplex PCR assay. Food samples were inoculated with STEC O157:H7 at concentrations of 10, 100 and 1000 CFU/mL. The two different concentration methods were compared based on 1) DNA yield 2) DNA purity and 3) detection limit and time of the target pathogen using a high-resolution melt cure multiplex real-time PCR assay for the detection of the major virulence genes, eaeA, stx1 and stx2, in E. coli O157:H7. DNA samples isolated and concentrated by SPRI showed better purity and yield compared to IMS cell concentration. A detection limit of 10 CFU was attained for the majority of inoculated food samples utilizing DNA samples concentrated using SPRI in just 4 h as opposed to 6 or more hours using IMS. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect low concentrations of E. coli O157:H7 in food samples that cannot be detected by conventional culture techniques.