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

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.

Rapid Detection of Escherichia coli O157:H7 by Loop-Mediated Isothermal Amplification Coupled with a Lateral Flow Assay Targeting the z3276 Genetic Marker

“…skilled personnel, both of which limit on-site testing. Over the last decade, several isothermal nucleic acid amplificationbased methods have been reported for the detection of this pathogen, for example, recombinant polymerase amplification (Rani et al 2021), rolling circle amplification (Du et al 2019), loop-mediated isothermal amplification (LAMP) (Ravan et al 2016), strand displacement amplification (Wu et al 2015), and crossing priming amplification (Zhou et al 2020). These isothermal amplifications offer great advantages including simple operation, a lack of specialized equipment, and visual detection of amplicon products.…”

“…It involves the amplification of six regions of the target with four pairs of primers under isothermal conditions (generally 60-65℃) in less than an hour (Notomi et al 2000). LAMP assays have been previously developed for the detection of E. coli O157:H7 (Rani et al 2021;Fei et al 2020;Zhang et al 2013), but these assays were not sufficiently specific to reliably detect this serotype. In response, Ravan and colleagues (Ravan et al 2016) developed an LAMP method that targeted the genetic marker z3276 for the detection of E. coli O157:H7, which had a detection limit of 10 CFU/reaction in pure bacterial culture.…”

Aptamer-based diagnostic and therapeutic approaches in animals: Current potential and challenges

“…There are many conventional methods to detect the presence of E. coli, and various assays have been developed to detect these strains at the herald of the diseases. The conventional method to detect E. coli usually takes two to three days ( Liu et al, 2020 , Rani et al, 2021 ). This method is not enough to characterize the bacteria, and additionally requires bacterial identification either by PCR ( Rajendhran and Gunasekaran, 2011 ), 16S rRNA sequencing, or by using biological probes, including antibodies and aptamers ( Gopinath et al, 2014 ).…”

Advanced Signal-Amplification Strategies for Paper-Based Analytical Devices: A Comprehensive Review

“…Conventionally, polymerase chain reaction (PCR) is the technique extensively used to rapidly process millions to billions of DNA samples, but it requires precise temperature control for reactions, and hence, to date has been unsuccessfully applied to PADs [149,150]. Alternatively, various signal amplification isothermal methods have been tried for PAD development, including rolling circle amplification (RCA) [147,[151][152][153][154][155], loop-mediated isothermal amplification (LAMP) [156][157][158][159][160][161][162][163][164][165][166], strand-displacement amplification (SDA) [167], recombinase polymerase amplification (RPA) [168][169][170][171], helicase dependent amplification (HDA) [172][173][174], and nucleic acid sequence-based amplification (NASBA) [175].…”

“…The results were obtained via the fluorescence signal of SYBR Gold dye and QuantiFluor dye with a very low LOD at 10 nm (3σ) and 25 nm (3σ), respectively, within 30 min at RT. In addition, RPA has been one of the most amplified methods for paper-based nucleic acid biosensor fabrication due to its outstanding ability to rapidly evaluate various gene targets at low temperature with superior primer specificity [170,171]. Based on these advantages, an RPA vertical flow paper microarray was developed for human adenoviral DNA detection by integrating it with colorimetric detection holding antibody-conjugated AuNPs (Figure 8B) [170].…”