Colorimetric Detection of the vanA Gene in Vancomycin-Resistant Enterococcus Using Loop-Mediated Isothermal Amplification with Hydroxy Naphthol Blue

Vancomycin-resistant enterococci (VRE), particularly vancomycin-resistant Enterococcus faecium, are important healthcare-associated pathogens, and vanA is a major acquired determinant of high-level glycopeptide resistance. The conventional polymerase chain reaction (PCR) method is reliable for molecular detection of vanA but requires thermal cycling and, in conventional formats, post-amplification analysis. In this study, we developed and evaluated a closed-tube colorimetric loop-mediated isothermal amplification assay using hydroxy naphthol blue (LAMP-HNB) for visual detection of vanA. A total of 130 Enterococcus isolates were tested, comprising 100 previously characterized in our published LAMP–lateral flow dipstick (LAMP-LFD) study (52 vanA-positive and 48 vanA-negative) and an additional 30 (13 vanA-positive and 17 vanA-negative), yielding 65 PCR-positive and 65 PCR-negative isolates overall. Reaction conditions were optimized sequentially, and the final LAMP-HNB protocol took place at 63 °C for 45 min with 4.0 mM MgSO4 and 5 μM HNB. The visual endpoint was confirmed by 2% agarose gel electrophoresis. LAMP-HNB correctly classified all 65 PCR-positive and all 65 PCR-negative isolates, yielding 100% sensitivity (65/65; exact 95% CI: 94.48–100%), 100% specificity (65/65; 95% CI: 94.48–100%), and 100% overall agreement (130/130; 95% CI: 97.20–100%). The limit of detection was 8.67 × 103 genome copies/µL (28.52 pg/µL) for LAMP-HNB compared with 8.67 × 104 genome copies/µL (285.2 pg/µL) for conventional PCR, representing a 10-fold lower DNA concentration endpoint under the experimental conditions employed. In the 100-isolate overlapping cohort, LAMP-HNB, the previously reported LAMP-LFD platform, and PCR were concordant. With its 45 min requirement for amplification after DNA preparation, the HNB format provides a rapid, closed-tube visual endpoint without a labeled probe, lateral flow strip, running buffer, or post-amplification hybridization step. These findings support LAMP-HNB as a promising molecular screening approach for vanA-positive enterococci, while larger, fully independent multicenter validation remains necessary before routine implementation.