Development of paper-based devices for detection of Vibrio parahaemolyticus DNA from loop-mediated isothermal amplification

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is a gram-negative, halophilic marine bacterium and marine seafood-borne pathogen causing gastrointestinal disorders to humans. In this work, colorimetric paper-based device was employed for the determination of V. parahaemolyticus DNA amplified by loop-mediated isothermal amplification (LAMP). LAMP was performed using a set of six specially designed primers which is specific to V. parahaemolyticus DNA. The optimal isothermal amplification was at 65°C for 45 min. After DNA amplifyed by LAMP, colorimetric assay was carried out on paper-based device for DNA determination using silver nanoparticles (AgNPs) as colorimetric agent. It was found that the color of AgNPs did not change in the presence of amplified DNA, whereas color of AgNPs changed from yellow to red in the presence of unamplified DNA. The results of the assay can be easily evaluated by naked eyes. The limit of detection (LOD) was found to be 11.15 CFU mL-1. Therefore, the developed method was used for the determination of V. parahaemolyticus DNA in seafood (shrimps and oysters). Comparison of the results obtained from this proposed method to those obtained by the plate count method (TCBS) and LAMP-gel electrophoresis proved that this developed method has good agreement with the conventional method in terms of accuracy for practical applications. Hence, LAMP-AgNPs on PADs serves as a rapid, inexpensive and simple assay without the need for complicated instrumentation.