Ward Linda N, Bej Asim K
Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170, USA.
Appl Environ Microbiol. 2006 Mar;72(3):2031-42. doi: 10.1128/AEM.72.3.2031-2042.2006.
We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.
我们开发了一种多重实时荧光定量PCR检测方法，在单个反应中使用四组基因特异性寡核苷酸引物和四种标记有不同荧光团的TaqMan探针，用于检测牡蛎中总副溶血性弧菌和致病性副溶血性弧菌，包括大流行的O3:K6血清型。副溶血性弧菌与食用生的或未煮熟的海鲜引起的食源性肠胃炎暴发有关，因此受到海鲜行业和消费者的关注。我们选择了针对耐热直接溶血素基因（tdh）和tdh相关溶血素基因（trh）的特异性引物和探针，据报道这些基因与该生物体的发病机制有关。此外，我们针对噬菌体f237的开放阅读框8（ORF8），它与新出现的致病性副溶血性弧菌O3:K6大流行血清型有关。使用不耐热溶血素基因（tlh）作为总副溶血性弧菌的检测靶点。发现组合的四基因座多重TaqMan PCR的灵敏度对于纯化的基因组DNA为200 pg，对于纯培养物为每毫升10(4) CFU。经过过夜富集后，可以检测到每克牡蛎组织匀浆中初始接种量为1 CFU的副溶血性弧菌，富集后浓度达到每毫升3.3x10(9) CFU。将该方法用于天然牡蛎检测，结果显示17/33的样本tlh呈阳性，4/33的样本tdh呈阳性。该检测方法能够特异性且灵敏地检测总副溶血性弧菌和致病性副溶血性弧菌，预计通过减少分析时间和无需进行多次检测，为传统检测方法提供一种快速可靠的替代方法。
