Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus

Abstract

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus's rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest. • The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success. • The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively. • The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance.