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Enhancement of loop mediated isothermal amplification s sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus
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Document Title
Enhancement of loop mediated isothermal amplification s sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus
Author
Lamalee A. Changsen C. Jaroenram W. Buates S.
Affiliations
Department of Microbiology Faculty of Science Mahidol University Bangkok 10400 Thailand; Bioengineering and Sensing Technology Research Team National Center for Genetic Engineering and Biotechnology (BIOTEC) National Science and Technology Development Agency (NSTDA) Pathum Thani 12120 Thailand
Type
Article
Source Title
MethodsX
ISSN
22150161
Year
2023
Volume
11
Open Access
All Open Access Gold Green
Publisher
Elsevier B.V.
DOI
10.1016/j.mex.2023.102328
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
License
CC BY
Rights
Authors
Publication Source
WOS