致病性副溶血性弧菌特异性三重PCR检测方法研究

Study on the Specific Triplex-PCR Method of Pathogenic Vibrio parahaemolyticus

[目的]建立同时检测致病性副溶血性弧菌gyrase、tdh、toxR基因的三重PCR快速检测方法。[方法]用3种基因的特异性引物分别对副溶血性弧菌ATCC33847的模板DNA进行单一扩增,找到各自引物最佳扩增条件;再用3种引物同步对模板DNA进行扩增,通过优化引物浓度、引物间比例以及退火温度,建立最佳扩增体系。[结果]在最佳三重PCR反应条件下,gyrase、tdh和toxR能同时扩增出清晰条带,大小分别为91、269和368 bp。[结论]该研究为致病性副溶血性弧菌的快速检测提供了一种新的技术方法。

[ Objective] To establish a rapid specific triplex-PCR method of pathogenic Vibrio parahaemolyticus by detecting gyrase, tdh and toxR genes simultaneously. [ Method] The genomic DNA of V. parahaemolyticus ATCC33847 was amplified by three pairs of specific primers individually, the optimum amplification condition for each pair of primers was determined. Through optimizing the concentration and ratio of primers and anneal temperature, a triplex-PCR method which can simultaneously amplify three target genes was established. [ Result] The bands exhibiting the sequences of 91, 269 and 368 bp were amplified by gyrase, tdh ,toxR respectively in an optimum triplex-PCR reaction. [ Conclusion] The triplex-PCR method provides a new technical mean for the rapid detection of pathogenic V. parahaemolyticus.