Multiple Cross Displacement Amplification Combined with Gold Nanoparticle-Based Lateral Flow Biosensor for Detection of Vibrio parahaemolyticus

Abstract

<i>Vibrio parahaemolyticus</i> (<i>V. parahaemolyticus</i>) is a marine seafood-borne pathogen causing severe illnesses in humans and aquatic animals. In the present study, multiple cross displacement amplification was combined with a lateral flow biosensor (MCDA-LFB) to detect the <i>toxR</i> gene of <i>V. parahaemolyticus</i> in DNA extracts from pure cultures and spiked oyster homogenates. Amplification was carried out at a constant temperature (62°C) for only 30 min, and amplification products were directly applied to the biosensor. The entire process, including oyster homogenate processing (30 min), isothermal amplification (30 min) and results indicating (∼2 min), could be completed within 65 min. Amplification product was detectable from as little as 10 fg of pure <i>V. parahaemolyticus</i> DNA and from approximately 4.2 × 10² CFU in 1 mL of oyster homogenate. No cross-reaction with other <i>Vibrio</i> species and with non-<i>Vibrio</i> species was observed. Therefore, the MCDA-LFB method established in the current report is suitable for the rapid screening of <i>V. parahaemolyticus</i> in clinical, food, and environmental samples.