The widespread emergence of transferable extensively drug-resistant (XDR) genes, including blaNDM and blaKPC for carbapenem resistance, mcr-1 for colistin resistance, and tet(X4) and tet(X6) for tigecycline resistance, in Enterobacteriaceae poses a major threat to public health. Thus, rapid on-site detection of these XDR genes is urgently needed. We developed a cascade system with a unitary polyethylene glycol (PEG) 200-enhanced recombinase polymerase amplification (RPA) as the core, combined with a modified Chelex-100 lysis method and a horseradish peroxidase (HRP)-catalyzed lateral flow immunoassay (LFIA) biosensor, to accurately detect these genes in Enterobacteriaceae. The conventional Chelex-100 lysis method was modified to allow in situ extraction of bacterial DNA in 20 min without requiring bulky high-speed centrifuges. Using PEG 200 increased the amplification efficiency of the RPA by 13%, and the HRP-catalyzed LFIA biosensor intensified the colorimetric signal of the test line. Following optimization, the sensitivity of the cascade system was <10 copies/μL with satisfactory specificity, allowing for highly sensitive detection of these XDR genes in Enterobacteriaceae. The complete detection procedure can be completed in less than 1 h without using large-scale instruments. This assay is conducive to rapid on-site visual detection of these XDR genes in Enterobacteriaceae in practical applications, thus providing better technical support for clinical surveillance of these genes and better treatment of XDR pathogens. IMPORTANCE Carbapenem, colistin, and tigecycline are considered the last resorts for treating severe bacterial infections caused by extensively drug-resistant (XDR) pathogens. A major threat to public health is the emergence and prevalence of transferable XDR genes in Enterobacteriaceae, such as blaNDM and blaKPC for carbapenem resistance, mcr-1 for colistin resistance, and tet(X4) and tet(X6) for tigecycline resistance. Therefore, it is imperative to develop rapid on-site methods to detect these XDR genes. In this study, we constructed a cascade system for detecting these genes based on PEG 200-enhanced recombinase polymerase amplification combined with a modified Chelex-100 lysis method and HRP-catalyzed lateral flow immunoassay. The current method is capable of detecting the above-mentioned XDR genes in situ with satisfactory specificity and sensitivity, which could provide technical support for the surveillance of these genes and provide medication recommendations for the treatment of relevant clinical infections.
Keywords: cascade detection system; colorimetric detection; isothermal amplification; multi-drug resistance genes; point-of-care testing.