A recombinase polymerase amplification-lateral flow assay (RPA-LFA) for Escherichia coli O157:H7 in food and beverages – Towards assured point-of-care diagnostics
Creators: name: Batra, Alka
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310703
subject: Microbial ecology
classificationCode: 4104
subject: Environmental management
classificationCode: 410404
subject: Environmental management

description: According to the World Health Organization (WHO), globally, 600 million people (1 in 10) fall sick every year after eating contaminated food, and over 2 billion people consume faecal-contaminated water. Escherichia coli O157:H7, a serotype of Shiga-toxin-producing E. coli has been listed as one of the most important food and waterborne pathogens which causes both acute and life-threatening chronic diseases such as haemorrhagic diarrhea, haemorrhagic colitis (HC), haemolytic-uremic syndrome (HUS) and haemolytic anaemia. Early and rapid detection of pathogens represent a key strategy to manage the spread of infection and related outbreaks. Despite showing good sensitivity and specificity, current methods are limited in terms of expense, time, complexity and trained labour requirement, making them unsuitable for on-site diagnosis. Recently, several isothermal DNA amplification techniques, including recombinase polymerase amplification (RPA) have been shown to be effective for the detection of various key pathogens; however, there is limited research on the development and assessment of RPA as a point-of-care (POC) detection tool for E. coli O157:H7 from food and beverages. Integrating RPA with a paper-based lateral flow assay (LFA) may overcome the limitation of current molecular assays which cannot be used for POC detection of E. coli O157:H7. The overarching aim of this thesis was to develop and validate an isothermal DNA amplification methodology for the POC detection of E. coli O157:H7 from food and beverages based on the use of RPA integrated with lateral flow technology.

With the aim of developing RPA-LFA for E. coli O157:H7 detection, Chapter 3 involved the use of PrimedRPA software to design primer pairs targeting three different genes (rfbE, fliC, stx2) of E. coli O157:H7 and subsequently assess their in-vitro amplified target DNA. Optimisation of time (5-30 min) and temperature (37-42 OC) for the primers targeting all 3 genes was carried out. Specificity and sensitivity assessment of the RPA-LFA was conducted through evaluation using drinking water, apple juice, and milk. Optimum amplification of E. coli O157:H7 DNA was achieved at 39 OC in 20 min irrespective of gene/primer types. The optimised RPA-LFA detected rfbE and stx2 gene targets with 100 % specificity; only 72.8% specificity was achieved with the fliC gene target when cross-reactivity was checked with other closely related bacteria including E. coli strains. The RPA was 100 times more sensitive than polymerase chain reaction (PCR); greatest sensitivity was observed using the stx2 gene, which could detect 100 ag DNA of E. coli O157:H7, some 10 - 100 times lower than the concentration detected using rfbE and fliC genes. The optimised RPA assay detected between 100 CFU/mL and 102 CFU/mL of E. coli O157:H7 using rfbE and stx2 gene targets. Two target candidates (rfbE and stx) were found more sensitive and specific for E. coli O157:H7 detection.

Chapter 4, continued the work with two selected gene targets stx2 and rfbE and aimed to use the RPA-LFA for the differential detection of dead and live E. coli O157:H7 and its assessment on viable but non-culturable (VBNC) E. coli O157:H7. To achieve this aim, an improved DNA intercalating dye called propidium monazide xx (PMAxx) was used in a range of 0-150 µM. RfbE gene amplification from dead E. coli O157:H7 was completely inhibited at 100 µM PMAxx; in contrast, stx2 gene amplification was only reduced to some extent with RPA-LFA. The sensitivity and selectivity of the PMAxx-RPA-LFA were found to be 102 CFU/mL and 100%, respectively when applied to dead cells, evaluated using viable but non-culturable (VBNC) E. coli O157:H7 cells from fresh milk, apple juice and drinking water. No effects of varying pH were seen on the PMAxx-RPA-LF assay. The results confirmed that the RPA-LFA can differentiate between live/dead E. coli O157:H7 in less than 40 min  if integrated with PMAxx and rfbE gene target. In addition, the assay can detect VBNC cells which can significantly overcome the limitation of culture-based and other molecular methods.

Chapter 5 aimed at improving the proposed qualitative RPA-LFA through the development of a quantitative assay. A more diverse range of food and beverage samples were used and RPA-LFA was compared with other DNA amplification-based detection approaches, namely polymerase chain reaction (PCR), real-time PCR (qPCR), and loop-mediated isothermal amplification (LAMP) for a better understanding of efficiencies in Affordability, Sensitivity, Specificity, User-friendliness, Rapid turnaround time, Equipment-involved, and Deliverability (ASSURED). This study also evaluated an in-house prepared, simple and cost-effective nitrocellulose paper-based dipstick technique for DNA extraction from E. coli O157:H7 spiked in different samples, with the aim to assess its efficacy in combination with the RPA assay, thereby overcoming the drawback of commercial DNA extraction kits. Moreover, the specificity of the RPA technology was cross validated in terms of the amplification of the target sequence amplicon. All 4 molecular methods used in this study were found to be 100 % specific using either commercial DNA extraction kit or the in-house extraction dipsticks. Quantitative RPA-LFA was found to be the most sensitive of the 4 molecular methods (p&lt;0.05) irrespective of the DNA extraction methodology. However, RPA-LFA was found the most suitable of the molecular assays for the POC detection of E. coli O157:H7, based on the ASSURED criteria. Amplicon readouts from Sanger sequencing validated the 100% specificity of RPA by showing 100% similarity with the expected in-silico amplicons using rfbE gene of E. coli O157:H7. The combination of nitrocellulose DNA extraction dipsticks with RPA-LFA increased the potential application of the proposed assay for the detection of E. coli O157:H7 contamination in remote settings areas. 

Overall, the research demonstrated that RPA-LFA targeting the rfbE gene could be a suitable rapid POC approach for the detection of E. coli O157:H7 contamination from food and beverages with minimal resource requirement in under 30 min on the same day of sampling. This research contributes to the understanding of the RPA technique and its potential application for the on-site detection of pathogens. The research conducted in this thesis produced encouraging results for E. coli O157:H7 contamination to support or categorise RPA-LFA as an ideal diagnostic test that has been coined as ASSURED by the WHO. Future work should include multiplexing of the assay to diagnose more than one foodborne or waterborne pathogen. The sensitivity of RPA technology could be increased with further research.