PreprintArticleVersion 1This version is not peer-reviewed
Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM)
Version 1
: Received: 9 July 2024 / Approved: 10 July 2024 / Online: 11 July 2024 (10:22:28 CEST)
How to cite:
Park, S. B.; Zhang, Y. Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM). Preprints2024, 2024070872. https://doi.org/10.20944/preprints202407.0872.v1
Park, S. B.; Zhang, Y. Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM). Preprints 2024, 2024070872. https://doi.org/10.20944/preprints202407.0872.v1
Park, S. B.; Zhang, Y. Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM). Preprints2024, 2024070872. https://doi.org/10.20944/preprints202407.0872.v1
APA Style
Park, S. B., & Zhang, Y. (2024). Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM). Preprints. https://doi.org/10.20944/preprints202407.0872.v1
Chicago/Turabian Style
Park, S. B. and Yan Zhang. 2024 "Improved Multiplex PCR Assay for Detection of tlh, trh, and tdh genes in Vibrio parahaemolyticus, Aligned with the U.S. FDA’s Bacteriological Analytical Manual (BAM)" Preprints. https://doi.org/10.20944/preprints202407.0872.v1
Abstract
Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)’s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM’s multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 minutes. Primer concentrations of 0.2 µM for trh, 0.2 µM for tlh, and 0.4 µM for tdh produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles (P < 0.05). This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio strains known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
