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

Yi Wang,Hui Li,Changyun Ye,Jianguo Xu,Dongxun Li,Yan Wang,Kewei Li

doi:10.3389/fmicb.2016.02047

Abstract

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.

Highlights

Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative halophilic bacterium that is widely distributed in marine, estuarine, and coastal environments (Letchumanan et al, 2014)
Multiple cross displacement amplification (MCDA)-lateral flow biosensors (LFB) for Detection of V. parahaemolyticus cases, the bacterium is responsible for ear infection, wound infection, or septicaemia that may be life-threatening to populations belonging to special at-risk groups, such as people with immune disorders or liver disease (Centers for Disease Control and Prevention, 2005)
The V. parahaemolyticus-MCDA primer set was a good candidate for development of the MCDA-LFB assay for V. parahaemolyticus detection

Summary

Introduction

Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative halophilic bacterium that is widely distributed in marine, estuarine, and coastal environments (Letchumanan et al, 2014). The organism is a major food-borne pathogen and frequently isolated from a variety of raw seafoods, such as shellfish, oysters, shrimp, crab, fish and lobster (Wang R. et al, 2015; Letchumanan et al, 2016). V. parahaemolyticus is able to cause acute gastroenteritis after the consumption of raw, undercooked or mishandled seafood (Su and Liu, 2007). MCDA-LFB for Detection of V. parahaemolyticus cases, the bacterium is responsible for ear infection, wound infection, or septicaemia that may be life-threatening to populations belonging to special at-risk groups, such as people with immune disorders or liver disease (Centers for Disease Control and Prevention, 2005). V. parahaemolyticus has the ability to cause serious illnesses in shellfish, fish and penaeid shrimp, resulting in significant losses in aquaculture industries (Tran et al, 2013)

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Discussion

Conclusion