Rapid and Highly Sensitive Detection of Toxigenic Vibrio cholerae Based on Recombinase-Aided Amplification Combining wit
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Rapid and Highly Sensitive Detection of Toxigenic Vibrio cholerae Based on Recombinase-Aided Amplification Combining with Lateral Flow Assay Weiwei Fang 1,2,3 & Yangyang Cai 1,2,3 & Li Zhu 4 & Hengliang Wang 4 & Ying Lu 1,2,3 Received: 24 February 2020 / Accepted: 1 November 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Vibrio cholerae . Recombinase-aided amplification . Lateral flow assay . Superparamagnetic nanoparticles
Introduction Vibrio cholerae, as a member of Vibrionaceae family, is frequently found in saline coastal waters and estuaries. Cholera toxin (CT) encoded by the ctxAB genes is the major virulence
* Hengliang Wang [email protected] * Ying Lu [email protected] 1
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, People’s Republic of China
2
Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, People’s Republic of China
3
Shanghai Engineering Research Center of Aquatic-Product Processing and Preservation, Shanghai 201306, People’s Republic of China
4
Beijing Institute of Biotechnology, Beijing 100071, People’s Republic of China
factor of V. cholerae (Awasthi et al. 2019). Cholera is an acute, watery diarrheal disease caused by V. cholerae, transmitted through contaminated water and food (Sayeed et al. 2
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