Recombinase polymerase amplification-lateral flow assay (RPA-LFA) as a rapid and sensitive test for Escherichia coli O157:H7 detection in food and beverage: A comparative study

Early and rapid detection of Escherichia coli O157:H7 is required to prevent food and water-borne outbreaks. Several diagnostic methods have been developed in recent decades; it is imperative to find the most sensitive and specific detection method to identify E. coli O157:H7 at the point-of-care (POC) settings. Recombinase polymerase amplification-lateral flow assay (RPA-LFA) has emerged with significant potential of POC application. Therefore, this study aimed to compare RPA-LFA with polymerase chain reaction (PCR), real-time PCR (qPCR), and loop-mediated amplification (LAMP) for use in the diagnosis of E. coli O157:H7. A total of 10 different food and beverages were collected, spiked, and tested, with all four detection methods combined with a lab-dependent (commercial kit) and field-deployable nitrocellulose paper-based DNA extraction method. A comparison of all four techniques based on important parameters such as Affordability, Sensitivity, Specificity, User-friendly, Rapid, Equipment-free, and Deliverability (ASSURED) was also conducted. In addition, the quantitative signal amplification by RPA-LFA was achieved through integration with a benchtop lateral flow strip reader. No significant difference was observed in terms of specificity (100% specific) among the detection techniques regardless of the DNA extraction method. In terms of sensitivity, for the commercial DNA extraction kit, the sensitivity of RPA-LFA was significantly higher compared to the other 3 techniques when applied to food and beverage samples (p < 0.05). The RPA-LFA was found the most sensitive method while LAMP was the least sensitive in detecting E. coli O157:H7 from solid matrices. For the field-deployable DNA extraction method, the sensitivity was also found to be significantly higher for RPA than qPCR, PCR, and LAMP (p < 0.05), irrespective of matrices type. The overall sensitivity of RPA-LFA was higher for beverages than foods. In summary, the inhouse developed nitrocellulose strips can be used to extract DNA from E. coli O157:H7 in under 1 min which can be amplified using RPA at 39 C-degrees for 15 min. Moreover, Sanger sequencing confirmed the specificity of the RPA technique for target amplification. The RPA-LFA, combined with an in-house developed field-deployable DNA extraction method showed potential as a POC detection tool to identify contamination in less than 25 min.