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

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