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. 2023 Apr 5;24(7):6784.
doi: 10.3390/ijms24076784.

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

Jin Tao  1 , Dejun Liu  1 , Jincheng Xiong  1 , Wenchong Shan  1 , Leina Dou  1 , Weishuai Zhai  1 , Yang Wang  1 , Jianzhong Shen  1 , Kai Wen  1
Affiliations

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

Jin Tao et al. Int J Mol Sci. .

Abstract

Recently, urinary tract infection (UTI) triggered by bacteria carrying 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. 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. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. 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 UTI treatment.

Keywords: Chelex-100; extensively drug-resistant genes; lateral flow immunoassay; recombinase polymerase amplification; urinary tract infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Scheme of the cascade detection system. (A) Schematic illustration of RPA, (B) HRP-catalyzed LFIA.
Figure 2
Figure 2
Feasibility of bacterial DNA extraction from uncultured bacterial-containing urine samples by refined Chelex-100 lysis method. (A) DNA concentration, (B) OD260/OD280 value, (C) OD260/OD230 value.
Figure 3
Figure 3
Comparison of the refined Chelex-100 lysis method with commercial kits applied in cultured bacterial-containing urine. (A) concentration of DNA, (B) OD260/OD280 value, (C) OD260/OD230 value.
Figure 4
Figure 4
Sensitivity for detecting pan-drug-resistant genes in urine samples. Linear regression and actual images. (A) mcr-1, (B) blaNDM, (C) blaKPC, (D) tet(X3), (E) tet(X4), (F) tet(X5), (G) tet(X6). NC, negative control.
Figure 5
Figure 5
Specificity analysis for pan-drug-resistant genes testing in urine samples. (A) mcr-1, (B) blaNDM, (C) blaKPC, (D) tet(X). NC, negative control.
See this image and copyright information in PMC

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