Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium

Abstract

The objective of this study was to establish a rapid visual diagnosis method for vancomycin-resistant Enterococcus faecium (VREFm) based on multienzyme isothermal rapid amplification (MIRA) combined with lateral flow strips (LFSs). The MIRA primers and probes were specifically designed to maintain the sequence of the VanA gene of VREFm. We optimized the reaction time and temperature and thoroughly assessed the specificity and sensitivity of the MIRA-LFS system. We also compared the MIRA-LFS method with the polymerase chain reaction (PCR) assay and the disc diffusion method. We then evaluated the MIRA-LFS assay for consistency testing and clinical application. The MIRA-LFS technique completed the amplification process within 30 min, and the results were observed on LFS. The method demonstrated high sensitivity, with a minimum detection limit of 1.066 CFU/µL for VREFm and exhibited specificity without cross-reactivity with other pathogenic bacteria. When applied to the detection of clinical samples, the method exhibited consistency with the PCR and agar dilution methods. The combined use of MIRA and LFS in this study facilitates simplifying the workflow for detecting VREFm, which is of great significance for rapidly detecting the enterococcal infections and preventing and controlling the nosocomial infections.

Importance: One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

Keywords: Enterococcus faecium; lateral flow strips; multienzyme isothermal rapid amplification; vancomycin-resistant.

FIG 1

The principle of MIRA-LFS.

FIG 2

Selection of the best primer-probe pair. (A) Agarose gel electrophoresis results of basic MIRA products. (B) MIRA-LFS results after base mismatch. (C) Sequence alignment between the VanA gene and the F/R/P.

FIG 3

Optimization of MIRA-LFS reaction conditions. (A) The MIRA assay was optimized for reaction time. (B) The MIRA assay was optimized for reaction temperature.

FIG 4

Specificity of the MIRA-LFS assay. (A) The MIRA-LFS assay was conducted using 10 standard pathogenic microbial strains. The ATCC IDs were labeled on the LFS. (B) The MIRA-LFS assay was conducted using 10 clinical VREfm. The sample numbers were labeled on the LFS.

FIG 5

Assay sensitivity of the MIRA-LFS system. (A) The MIRA-LFS assay was conducted using different concentrations of VRFfm. The concentrations were labeled on the LFS. (B) The LOD of the MIRA-LFS was estimated using a probit regression model based on 10 independent replicates of each serial dilution.