Rapid and Highly Sensitive Detection of Toxigenic Vibrio cholerae Based on Recombinase-Aided Amplification Combining with Lateral Flow Assay

Abstract

Vibrio cholerae is an important food-borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. Rapid diagnosis of cholera infection is important to avoid potential spread of disease. In this study, recombinase-aided amplification (RAA) primers labeled with biotin and fluorescein isothiocyanate (FITC) at 5′ end were prepared for the ctxA pathogenic gene of V. cholerae. RAA amplicons obtained at 37 °C isothermal conditions for 15 min were firstly preincubated with the antibody against FITC, subsequently with streptavidin-modified superparamagnetic nanoparticles (SA-SPMNPs), to form a DNA amplicons-FITC antibody-SA-SPMNPs complex. The complex could be captured by the secondary antibody on test line of lateral flow test strip (LFTS), and the residual streptavidin on SA-SMMNPs could be captured by the biotin-BSA on the control line of LFTS. Thus, a sensitive quantitative detection was achieved by the magnetic signal of test line of LFTS through a magnetic assay instrument. The RAA amplicons from various bacteria (n = 25) obtained by a thermostatic water bath for 15 min at 37 °C were used as samples of lateral flow assay (LFA). No positive reaction was observed besides of pathogenic V. cholerae. A total of 100 CFU/ml V. cholerae in shrimp samples could be detected by a simple naked eye observation within 50 min including the sample pretreatment. Quantitative LOD 46 CFU/ml V. cholerae in shrimp was achieved, which was more than 10 times lower than the current industry pathogenic range. The novel RAA-LFA shows a potential application in the point-of-care diagnosis or detection of V. cholerae.