Development of concentration and detection methods for the detection of extended-spectrum beta-lactamase producing bacterial pathogens in foods

Abstract

Currently used conventional methods for detection of bacteria in foods are time consuming and labor intensive. Therefore, there is an increasing demand for a robust, rapid, and cost-effective method for the detection of foodborne pathogens. Multiplex high-resolution melt-curve (HRM) real-time polymerase chain reaction (PCR) assays can be used as an effective technique for rapid and simultaneous detection of foodborne pathogens. In this study, two novel multiplex HRM real-time PCR assays were developed for the simultaneous detection of virulence and extended spectrum β-lactamase (ESBL) genes in Salmonella. A total of six virulence and three ESBL genes, which included hilA, fimH, sipA, invA, fimA, stn, blaTEM, blaSHV and blaCMY, were detected by the two assays. The application of these assays in detection of Salmonella in foods was tested by artificially spiking 27 different food samples using three different spike levels of Salmonella, viz., 10, 102 and 103 CFU/mL. These assays were able to detect as low as 10 CFU/mL in 25 g of bacterial cells within 10 h of enrichment. In addition, a novel multiplex HRM real-time PCR assay was developed for the simultaneous detection of Shiga toxin-producing Escherichia coli (STEC) and ESBL-producing E. coli in beef products. The assay targeted stx1, stx2, eaeA, uidA and blaCTX-M genes. A detection limit of 10 CFU in 325 g of beef was obtained within 8 h of enrichment. Moreover, a novel, rapid and efficient technique for upstream bacterial DNA purification and concentration from food matrices was developed. This was accomplished by creating an upstream bacterial DNA isolation and concentration approach using solid phase reversible immobilization (SPRI) paramagnetic beads. By comparing the DNA content, purity, and limit of detection utilizing multiplex HRM PCR created to detect E. coli O157:H7, the effectiveness of the developed approach was assessed with the commercially available immunomagnetic separation (IMS) method. The DNA samples isolated and concentrated using the SPRI-based concentration method showed better purity and yield compared to the IMS-based method. Additionally, a detection limit of 10 CFU was attained in the majority of the spiked food samples utilizing DNA samples concentrated using the SPRI-based method in just 4 h as opposed to 6 or more hours using the IMS-based method. The multiplex assay was found to be specific for the detection of E. coli O157:H7. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect E. coli O157:H7 that are present in food samples in low quantities but cannot be detected by conventional culture techniques.