Rapid detection of extended spectrum β-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis

• DOI: 10.1371/journal.pone.0248536
• Publication Date: 2021-03-15T17:25:02Z
• Authors: Sitthichai Kanokudom; Thachaporn Assawa...; Yukihiro Akeda; Panan Ratthawongj...; Rungtip Chuanchuen; Nuntaree Chaichan...
• Keywords: 0301 basic medicine; Molecular biology; Science; Multiplex polymerase chain reaction; Clinical Biochemistry; Biomedical Engineering; FOS: Basic medicine; FOS: Medical engineering; Microbiology; Gene; beta-Lactamases; Global Challenge of Antibiotic Resistance in Bacteria; Microbial Identification and Diagnosis; Detection limit; 03 medical and health sciences; Engineering; Enterobacteriaceae; Biochemistry, Genetics and Molecular Biology; Virology; Escherichia coli; Genetics; Cecum; Biology; Multiplex; Chromatography; ESKAPE Pathogens; Escherichia coli Proteins; Q; R; Life Sciences; Amplicon; Recombination; Polymerase chain reaction; 3. Good health; Chemistry; FOS: Biological sciences; Physical Sciences; Recombinase; Food Microbiology; Pork Meat; Medicine; Molecular Medicine; Paper-Based Diagnostic Devices; Recombinase Polymerase Amplification; Multiplex Polymerase Chain Reaction; Research Article

The emergence and dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a global health issue. Food-producing animals, including pigs, are significant reservoirs of antimicrobial resistance (AMR), which can be transmitted to humans. Thus, the rapid detection of ESBLs is required for efficient epidemiological control and treatment. In this study, multiplex recombinase polymerase amplification (RPA) combined with a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS), as a lateral flow strip assay (LFA), was established for the rapid and simultaneous detection of multiple bla genes in a single reaction. Visible blue lines, indicating the presence of the blaCTX-M, blaSHV, and blaOXA genes, were observed within 10 min by the naked eye. The limit of detection of all three genes was 2.5 ng/25 μL, and no cross-reactivity with seven commensal aerobic bacteria was observed. A total of 93.9% (92/98) and 96% (48/50) of the E. coli isolates from pork meat and fecal samples, respectively, expressed an ESBL-producing phenotype. Nucleotide sequencing of the PCR amplicons showed that blaCTX-M was the most prevalent type (91.3–95.83%), of which the main form was blaCTX-M-55. The sensitivity and specificity of the RPA-LFA were 99.2% and 100%, respectively, and were in almost perfect agreement (κ = 0.949–1.000) with the results from PCR sequencing. Thus, the RPA-LFA is a promising tool for rapid and equipment-free ESBL detection and may facilitate clinical diagnosis in human and veterinary medicine, as well as AMR monitoring and surveillance.