Nanoparticle-based lateral flow biosensor combined with multiple cross displacement amplification for rapid, visual and sensitive detection of Vibrio cholerae

Abstract

Vibrio cholerae is an important human pathogen that is responsible for cholera, a severe acute watery diarrhea. In the current study, a multiple cross displacement amplification (MCDA) coupled with amplicon detection by chromatographic lateral flow biosensor (LFB) method (MCDA-LFB) was successfully established and evaluated for the identification of V. cholerae. A set of 10 primers was designed specifically to recognize 10 different regions of the V. cholerae-specific gene ompW. The optimized time and temperature conditions for the MCDA were 30 min and 63°C, respectively. The MCDA-LFB assay correctly identified 31 strains of V. cholerae but did not detect 13 non-cholerae Vibrio strains and 30 non-Vibrio strains. The sensitivity of MCDA-LFB for target pathogen detection in pure culture was 10 fg per reaction. In the case of spiked shrimp samples without enrichment, the limit of detection was 4.1 CFUs per reaction or equivalent to 4.1 × 10² CFU g⁻¹. The whole process, including shrimp homogenates processing (30 min), MCDA reaction (30 min) and results reporting (2 min), could be finished within 65 min. These results show that this assay is suitable for the rapid, sensitive and specific detection of V. cholerae in food, environmental and clinical samples.