toxR基因作为荧光定量PCR靶基因设计TaqMan探针快速检测副溶血弧菌

Rapid Detection of Vibrio parahaemolyticus by TaqMan-based Real-time PCR Assay Targeting the toxR Gene

摘要:

本研究以toxR基因为靶基因, 通过优化反应条件建立了快速检测副溶血弧菌的TaqMan实时荧光PCR方法。特异性试验表明, 该方法能选择性检测副溶血弧菌, 而与金黄色葡萄球菌、沙门氏菌、单增李斯特杆菌等多种常见的食源性病原菌没有交叉反应; 灵敏度试验表明, 该方法最少可检测到25个拷贝的toxR基因重组质粒, 对纯培养物和模拟食品样品直接检测的灵敏度分别为21 cfu/mL和210 cfu/g; 重复性试验表明, 同一样品于试验内及试验间的变异系数分别为0.9%和1.3%; 所制作的标准曲线在2.5×101～2.5×106拷贝数之间有较好的线性关系, 能对副溶血弧菌进行准确的定量分析。结果表明, 本研究所建立的副溶血弧菌实时荧光PCR检测方法具有特异性好、灵敏度高、重复性好的特点, 能进行定量检测, 而且检测时间从核酸抽提到出实验结果仅需要3 h, 是快速检测副溶血弧菌的有效手段。

Abstract:

We designed a pair of specific primers and a TaqMan fluorescent probe targeting the toxR gene of Vibrio parahaemolyticus (VP). After optimizing the conditions, the specialty, sensitivity and reproducibility of the detection method were evaluated. Results: (1) the developed real-time PCR assay protocol detected only VP and was not affected by other normal food pathogens such as Staphylococcus aureus, Salmonela, Listeria monocytogenes. (2) the limit of detection was 25 copies of toxR gene in the detected samples, and the sensitivity of pure cultures and simulated food samples was 21cfu/ml and 210cfu /g. (3) the developed protocol of real-time PCR assay had a high reproducibility, and the sample’s variation was 0.9% and 1.3% within the same sample and between tests. (4) the standard curve had a good linearity when the gene quantity was between 2.5×101 and 2.5×106 copies. The developed detection assay targeting the toxR gene can quantitatively detect VP in only 3 hours, and thus is an efficacious method for the detection of Vibrio parahaemolyticus.