Park Seong Bin, Zhang Yan
Experimental Seafood Processing Laboratory, Coastal Research & Extension Center, Mississippi State University, Pascagoula, MS 39567, USA.
Pathogens. 2024 Sep 7;13(9):774. doi: 10.3390/pathogens13090774.
is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s () recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin () gene and the pathogenic thermostable-related hemolysin () and thermostable-direct hemolysin ( genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the (486 bp) and (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the gene amplicon (270 bp). The present study aimed to improve the 's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the gene (369 bp) alongside the existing primers for the and 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 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 .
是一种重要的食源细菌，食用受污染的海鲜后会引发严重的肠胃炎。为了鉴定并确定其致病性，美国食品药品监督管理局（FDA）的（）推荐了一种多重聚合酶链反应（PCR）方案，以同时检测物种特异性不耐热溶血素（）基因以及致病性耐热相关溶血素（）和耐热直接溶血素（基因）。然而，该检测方法存在两个局限性：由于不耐热溶血素（486 bp）和耐热直接溶血素（450 bp）基因的扩增子大小高度相似，难以分离；且耐热相关溶血素基因扩增子（270 bp）显示出较弱的条带。本研究旨在通过分离强度相似的三个扩增子来改进的多重PCR检测方法。除了用于和基因的现有引物外，还针对耐热相关溶血素基因（369 bp）应用了一组新引物。通过在2%的三羟甲基氨基甲烷-硼酸-乙二胺四乙酸（TBE）琼脂糖凝胶上进行电泳，在45分钟内有效分离了三个基因的扩增子。在35个PCR循环的各种引物浓度组合中，三个基因0.25 μM 的引物浓度产生了大量的扩增子。该检测方法的检测限为10 pg细菌DNA，显示出其高灵敏度。它未显示来自已知的九种人类病原体或18种有充分文献记载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，并为检测的三个基因提供更可靠的方法。
