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

Vibrio parahaemolyticus (V. parahaemolyticus) 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 toxR gene of V. parahaemolyticus 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 V. parahaemolyticus DNA and from approximately 4.2 × 102 CFU in 1 mL of oyster homogenate. No cross-reaction with other Vibrio species and with non-Vibrio species was observed. Therefore, the MCDA-LFB method established in the current report is suitable for the rapid screening of V. parahaemolyticus in clinical, food, and environmental samples.

Protocol publication

[…] The MCDA primer pairs (F1, F2, CP1, CP2, C1, C2, D1, D2, R1 and R2) were designed using PrimerExplorer V4 (Eiken Chemical, Japan) and primer software PRIMER PREMIER 5.0. All primers were analyzed for hairpin structures and hybrids using the Integrated DNA Technologies design tools. Blast analysis was used to verify that the MCDA primers were specific for V. parahaemolyticus. The CP1 (C1+P1) and C1 primers were labeled at their 5′ end with biotin and fluorescein isothiocyanate (FITC), respectively. The sequences, positions and modifications of the primer pairs are displayed in Figure and Table . All of the oligomers were synthesized and purified by TsingKe Biotech Co., Ltd. (Beijing, China) at HPLC purification grade. […]