Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters
Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters
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DOI：
10.1128/aem.00460-07
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
DePaola, Angelo
中科院分区：
文献类型：
--
作者：
Nordstrom, Jessica L.;Vickery, Michael C. L.;DePaola, Angelo
副溶血性弧菌是一种河口细菌，是美国贝类相关细菌性胃肠炎病例的主要原因。本实验室建立了同时检测副溶血弧菌不耐热溶血素(Tlh)、耐热直接溶血素(Tdh)和耐热相关溶血素(Trh)基因的实时多重聚合酶链式反应方法。Tlh基因是一个物种特异性标记，而tdh和trh基因是致病标记。采用内部扩增对照(IAC)来确保聚合酶链式反应的完整性和消除假阴性报告。该方法对代表8种细菌的150株细菌进行了特异性检测。只有具有适当靶基因的副溶血性弧菌才能产生荧光信号，除了3株霍利氏弧菌产生的晚期TdH信号外。多重试验检测致病性副溶血弧菌与副溶血弧菌总菌落的<；10 cfu/反应。在阿拉斯加牡蛎的环境调查中，以最可能数字的形式使用实时PCR分析，并将其结果与标准的副溶血性弧菌分离方法进行比较。IAC偶尔会被牡蛎基质抑制，这通常对应于副溶血性弧菌靶标的阴性结果。副溶血性弧菌tlh、tdh和trh分别在44、44和52%的牡蛎样品中检出。副溶血性弧菌的分离率为33%，TdH(+)和trh(+)菌株的分离率分别为19%和26%。这些结果证明了实时聚合酶链式反应在环境调查中的实用性，以及它可能应用于暴发调查以检测总的和致病的副溶血性弧菌。
Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a speciesspecific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against > 150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected < 10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of > 10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.