Journal
FOOD ANALYTICAL METHODS
Volume 14, Issue 4, Pages 687-696Publisher
SPRINGER
DOI: 10.1007/s12161-020-01909-x
Keywords
Vibrio cholerae; Recombinase-aided amplification; Lateral flow assay; Superparamagnetic nanoparticles
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Funding
- National Science and Technology Major Project of China [2018ZX10101003]
- State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science) [SKLPBS1832]
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Vibrio cholerae is a significant foodborne pathogen that causes human diseases, and a rapid and sensitive detection method using recombinase-aided amplification (RAA) and lateral flow assay (LFA) has been developed. This method allows for the detection of V. cholerae within 50 minutes, with a quantitative limit of detection (LOD) of 46 CFU/ml, which is more than 10 times lower than the current industry standard. The novel RAA-LFA technique has potential applications in point-of-care diagnosis or detection of V. cholerae.
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 degrees 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 degrees 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.
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