Portable Differential Detection of CTX-M ESBL Gene Variants, bla _CTX-M-1 and bla _CTX-M-15 , from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification

Abstract

CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control.

Discriminating bovine mastitis pathogens by combining loop-mediated isothermal amplification and amplicon-binding split trehalase assay

“…The extended loop primers of the initialized three primer sets were critical to obtain endpoint concatemer products containing large amounts of the target of our fusion protein reagents, because the loop primers simultaneously introduced the specific sequences and boosted the LAMP reaction. Interestingly, the presented research is one of very few studies relying on modifying LAMP loop primers for enhanced amplicon detection ( 37 – 39 ).…”

Detection of Extended-spectrum β-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection

“…Several assays for genotyping ESBL genes, exist: (1) Kanokudom et al, 14 developed a naked-eye rapid multiplex RPA assay for detecting bla CTX-M , bla OXA , and bla SHV in pork E. coli isolates. Amplicons were visualized on a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS, a commercial lateral flow assay strip); (2) Higgins et al, 36 set up a portable loop-primer endonuclease cleavage-loop-mediated isothermal amplification (loop-primer endonuclease cleavage-LAMP) assay for detecting bla CTX-M-1 and bla CTX-M-15 in porcine fecal E. coli isolates; and (3) Wang et al developed a probe-based real-time PCR assay for detecting bla CTX-M , bla TEM , and bla SHV in broiler chicken E. coli isolates 37 .…”

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection

“…Therefore, the facility for the probe-based LAMP methods is more likely to be more costly than the traditional LAMP methods. It is worth noting that some portable fluorescence analyzers and isothermal fluorimeters with minimal laboratory resources are currently available or under development (Supplementary Table S1) [70][71][72][73][74][75]. Furthermore, end-point fluorescence detection requires much less instrumentation than real-time monitoring.…”

“…Second, the results of the probe-based LAMP methods can be real-time monitored using a real-time (thermal or isothermal) fluorimeter or be observed at the end-point using a regular fluorimeter [70,71,73]. A small, portable, cheap, and battery-powered isothermal fluorimeter should be developed.…”