Rapid On-Site Detection of Extensively Drug-Resistant Genes in Enterobacteriaceae via Enhanced Recombinase Polymerase Amplification and Lateral Flow Biosensor

Abstract

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 bla NDM and bla KPC for carbapenem resistance, mcr-1 for colistin resistance, and tet (X4) and tet (X6) for tigecycline resistance.

MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples

“…Due to the RPA reaction circumstances used in this study, for the remaining above-mentioned pan-drug-resistant genes, except for tet (X) determined in our previous study, only the conditions for the detection of tet (X) were determined [ 12 ]. According to the results, the final fluorescence intensity was greatest at 41 °C after 15 min ( Figure S3, Table S3 ).…”

“…Consequently, a method for the rapid extraction of DNA from urine samples appropriate for on-site use is warranted. Previously, we used a lateral flow immunoassay (LFIA), a strip-based platform using antigen–antibody specific binding, to allow rapid in-field detection of RPA amplification products [ 12 ].…”

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

“…The procedure of PEG 200 enhanced exo-RPA was performed based on our previous study, and the detailed information was displayed in the Supplementary Information. 12 2.6 HRP-catalyzed lateral ow immunoassay…”

“…Previously, we constructed a procedure for extracting DNA from bacteria using the modi ed Chelex-100 lysis method. 12,13 In this study, we attempt to use the approach to extract bacterial DNA directly from urine samples. The speci c work ow is as follows:…”

“…Previously, we used a lateral ow immunoassay (LFIA), a stripbased platform using antigen-antibody speci c binding, to allow rapid in-eld detection of RPA ampli cation products. 12 Therefore, for POC testing the pan-drug resistant gene in UTI samples, an MCL-PRPA-HLFIA cascade detection system was designed based on previously developed PEG 200 enhanced RPA, along with a re ned Chelex-100 lysis technique and HRP-catalyzed LFIA. Within an hour, the system performs POC detection of the genes with high sensitivity and speci city, providing appropriate dosing guidance for the treatment of clinical UTI.…”