Evaluation and comparison of recombinase polymerase amplification coupled with lateral-flow bioassay for Escherichia coli O157:H7 detection using different genes

Alka Rani,Andrew S Ball,Vivek B Ravindran,Nagalakshmi Haleyur,Esmaeil Shahsavari,Aravind Surapaneni,Nitin Mantri

doi:10.1038/s41598-021-81312-6

Abstract

Shiga toxin-producing Escherichia coli serotype O157:H7 is a food and waterborne zoonotic pathogen causing gastroenteritis in humans. Rapid and simple detection in water and food is imperative to control its spread. However, traditional microbial detection approaches are time-consuming, expensive and complex to operate at the point-of-care without professional training. We present a rapid, simple, sensitive, specific and portable method for detection of E. coli O157:H7 in drinking water, apple juice and milk. We evaluated the effect of gene selection in detecting E. coli O157:H7 using recombinase polymerase amplification coupled with a lateral flow assay using rfbE, fliC and stx gene targets. As low as 100 ag and 1 fg DNA, 4–5 CFU/mL and 101 CFU/mL of E. coli O157:H7 was detected using the stx and rfbE gene targets respectively with 100% specificity, whilst the detection limit was 10 fg DNA and 102 CFU/mL for the fliC gene target, with 72.8% specificity. The RPA-LFA can be completed within 8 min at temperatures between 37 and 42 °C with reduced handling and simple equipment requirements. The test threshold amplification of the target was achieved in 5–30 min of incubation. In conclusion, RPA-LFA represents a potential rapid and effective alternative to conventional methods for the monitoring of E. coli O157:H7 in food and water.

Highlights

Shiga toxin-producing Escherichia coli serotype O157:H7 is a food and waterborne zoonotic pathogen causing gastroenteritis in humans
According to the World Health Organization (WHO), in 2005, 1.8 million people died from diarrheal diseases, traced to the consumption of food contaminated with pathogens including E. coli O157:H73
The Recombinase polymerase amplification (RPA)-AGE results confirmed the amplification of target E. coli O157:H7 with rfbE F1/R1, rfbE F2/R2, rfbE F3/R3, rfbE F4/R4, fliC F2/R2, fliC F3/R3, fliC F4/R4, Eco stx[2] F1/R1, Eco stx F2/R2 primer sets; no amplification was attained with fliC F1/R, stx[1] F1/R1 and stx[1] F2/R21

Summary

Introduction

Shiga toxin-producing Escherichia coli serotype O157:H7 is a food and waterborne zoonotic pathogen causing gastroenteritis in humans. Molecular assays such as polymerase chain reaction (PCR)[6] and pulse field gel electrophoresis (PFGE)[7] have been developed for the identification of E. coli O157:H7 These methods overcome the limitation of culture based methods, having good sensitivity and specificity; these techniques require sophisticated devices, trained personnel and are generally expensive. Lateral flow (LF) paper-based microfluidics (LFPBM) is an emerging detection device that fulfils the criteria of an ideal POC diagnostic tool given by the World Health Organization (WHO) as ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Deliverable[10]. The principle of these LF devices is the capillary action of fluids through 3-dimensional porous microstructures. The specificity was found satisfactory and the detection limit was 1 × 1 04 CFU/mL, the complete assay takes 3–4 h and requires multiple antibodies, making the assay 3–4 times more expensive than traditional culture based and molecular methods[13]

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