Development of closed dumbbell mediated isothermal amplification assay for rapid and on-site detection of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus), a gram-negative food-borne pathogen, exists widely in the global marine environment and poses a grave threat to human. Thus, simple, accurate and rapid detection of V. parahaemolyticus is vital for food safety and effective treatment. However, existing methods are difficult to meet the requirements of detection limit, sensitivity and specificity for rapid V. parahaemolyticus diagnosis. The aim of this study was to develop a nucleic acid-based assay by the newly-established closed dumbbell mediated isothermal amplification (CDA) to detect V. parahaemolyticus with high sensitivity and specificity. The suitable inner primers of CDA were designed and screened to specially amplify the conserved target gene of V. parahaemolyticus. Besides, outer primers were designed to accelerate and optimize the CDA reaction process of V. parahaemolyticus gene amplification. And the detection limit of the developed CDA assay for V. parahaemolyticus was 1 copy/μL, which was 10-fold lower than real-time quantitative PCR. Moreover, the CDA assay coupled with Hydroxy naphthol blue (HNB) indicator realized endpoint judgement by naked eye, which would be helpful for onsite diagnosis. In conclusion, the developed real-time fluorescence and visual V. parahaemolyticus CDA assays were suitable for point of care test, holding great potential in food security, environment monitoring and clinical testing.