Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA’s Bacteriological Analytical Manual (BAM)

IF 3.3 3区医学 Q2 MICROBIOLOGY

Pathogens Pub Date : 2024-09-07 DOI:10.3390/pathogens13090774

Seong Bin Park, Yan Zhang

{"title":"Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA’s Bacteriological Analytical Manual (BAM)","authors":"Seong Bin Park, Yan Zhang","doi":"10.3390/pathogens13090774","DOIUrl":null,"url":null,"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 highly 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 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species 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.","PeriodicalId":19758,"journal":{"name":"Pathogens","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Pathogens","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3390/pathogens13090774","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MICROBIOLOGY","Score":null,"Total":0}

引用次数: 0

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 highly 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 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species 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.

查看原文本刊更多论文

用于检测副溶血性弧菌中 tlh、trh 和 tdh 基因的创新型多重 PCR 分析法，参照美国 FDA 的《细菌分析手册》（BAM）

副溶血性弧菌是一种重要的食源性细菌，食用受污染的海产品后会引起严重的肠胃炎。为鉴别副溶血性弧菌并确定其致病性，美国食品药品管理局（FDA）的《细菌分析手册》（BAM）建议采用多重聚合酶链反应（PCR）方案，同时检测物种特异性热溶血素（tlh）基因以及致病性热稳定相关溶血素（trh）和热稳定直接溶血素（tdh）基因。然而，这种检测方法有两个局限性：一是由于 trh（486 bp）和 tlh（450 bp）基因的扩增子大小非常相似，因此很难将其分开；二是 tdh 基因扩增子（270 bp）的条带较弱。本研究旨在通过分离强度相似的三个扩增子来改进 BAM 的多重 PCR 检测。在使用现有的 trh 和 tdh 基因引物的同时，还使用了一套新的 tlh 基因引物（369 bp）。在 2% 三硼酸-EDTA（TBE）琼脂糖凝胶上电泳 45 分钟，三个基因的扩增子被有效分离。三个基因的引物浓度为 0.25 µM，在 35 个 PCR 循环的不同引物浓度组合中产生了大量的扩增子。该检测方法的细菌 DNA 检测限为 10 pg，显示了其高灵敏度。它没有检测到 9 种已知为人类病原体的弧菌或 18 种证据确凿的食源性病原体的扩增子。因此，目前的多重 PCR 方案有助于克服现有检测方法的局限性，为检测副溶血性弧菌的三个基因提供更可靠的方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Pathogens Medicine-Immunology and Allergy

CiteScore

6.40

自引率

8.10%

发文量

1285

审稿时长

17.75 days

期刊介绍： Pathogens (ISSN 2076-0817) publishes reviews, regular research papers and short notes on all aspects of pathogens and pathogen-host interactions. There is no restriction on the length of the papers. Our aim is to encourage scientists to publish their experimental and theoretical research in as much detail as possible. Full experimental and/or methodical details must be provided for research articles.

文献相关原料

公司名称	产品信息	采购帮参考价格