以16S和16S—23S rDNA间区为靶区建立副溶血弧菌(Vibrio parahaemolyticus) PCR快速检测技术

提要应用BLAST程序和DNAstar软件,比较分析了5株副溶血弧菌和其他细菌的16S—23S rDNA间区序列,选取IGSIA作为扩增靶区,结合相邻的16S rDNA序列,设计合成了一对特异性引物,通过优化扩增条件,建立了快速检测副溶血弧菌的PCR方法,对其特异性和敏感性进行了探讨,并初步进行了应用研究。结果表明,新建的PCR方法能特异性地扩增出大小为306bp和552bp的2条带,分别对应于副溶血弧菌的IGS0和IGSIA,检测灵敏度为5.6×102CFU/ml,半个工作日即可得到准确的结果,能有效检测出在杂色鲍、凡纳滨对虾和各种水质中的副溶血弧菌,适用于海洋水产动物副溶血弧菌病的早期快速诊断、水产品的检疫及水质环境的监测。

RAPID PCR DETECTION FOR VIBRIO PARAHAEMOLYTICUS USING 16S AND 16S-23S rDNA AS TARGET

Using software BLAST and DNAstar, the 16S-23S rDNA intergenic spacer (IGS) sequences of 5 strains of Vibrio parahaemolyticus were analyzed with those of other bacteria available in GenBank. Based on the results from multiple sequence alignment of representative IGS^IA type and its flanking 16S rDNA, a pair of specific PCR primers for V. parahaemolyticus was successfully designed and synthesized. A new PCR technique was developed by optimizing the amplification condition. The species-specificity and sensitivity of this new method were tested and used to detect V. parahaemolyticus in experimentally infected abalone, tissue of cultured shrimps, and water samples. The results show that two PCR products (316 and 552bp, corresponding to the IGS⁰ and IGS^IA, respectively) were co-amplified in all tested V. parahaemolyticus strains. Detection limit of this method was 5.6×10² CFU/ml, and the detection could be finished within a half day. This new PCR method may have a potential use in the development of diagnosis methods for mariculture maintenance, seafood quarantine, and the water quality monitoring.