Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M

Background: Salmonella enterica and Escherichia coli are common foodborne pathogens of global concern, particularly due to their antimicrobial resistance, notably to cephalosporins. Objective: This study aimed to evaluate a polymerase chain reaction-based lateral flow strip (PCR-LFS) assay for the detection of Salmonella spp. and E. coli harboring the blaCTX-M gene, which confers resistance to third-generation cephalosporins. Methods: Two duplex PCRs (dPCR) were established to detect E. coli-harboring blaCTX-M (set 1) and Salmonella-harboring blaCTX-M (set 2). 600 bacterial isolates and raw pork mince spiked with blaCTX-M-harboring E. coli and Salmonella were used to evaluated. Results: Both dPCR assays successfully detected blaCTX-M-positive E. coli or Salmonella strains, while strains lacking the gene showed no amplification. Non-E. coli and non-Salmonella strains were PCR-negative unless they carried blaCTX-M. The dPCR-LFS showed 100% validity including accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for both E. coli or Salmonella spp. harboring or lacking blaCTX-M. The assay accurately detected target strains without cross-reactivity with other bacteria or antimicrobial resistance genes. Cohen’s Kappa coefficient indicated perfect agreement (κ = 1), reflecting the high reliability of the dPCR-LFS. The assay could detect as low as 25 CFU/mL for blaCTX-M-positive E. coli and 40 CFU/mL for blaCTX-M-positive Salmonella in spiked raw pork mince. Conclusions: This assay is rapid, easy to interpret, and suitable for large-scale screening in surveillance programs.