MC-PRPA-HLFIA cascade detection system for point-of-care testing pan-drug resistant genes in urinary tract infection samples

Abstract

Abstract Background Recently, urinary tract infection (UTI) triggered by bacteria carrying the pan-drug resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. We intend to develop a rapid on-site assay, which is capable of direct detection of pan-drug resistant genes in clinical UTI samples. Methods Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. Results The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 minutes without high-speed centrifugation or pre-incubating urine samples. Following optimization, the cascade detection system achieved a LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. Conclusions It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug resistant genes in clinical UTI samples and provides the necessary medication guidance for the UTI treatment.