Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithin-dependent hemolysin gene.

Abstract

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg(2+)), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg(2+)) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1-2 hours, with a minimum bacterial density of 10 CFU.mL(-1) and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.