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. ? 2023
