节瘤拟杆菌PCR检测方法的建立

Establishment of a PCR Technique to Detection of I,II Groups of D.nodosus in Footrot in Ruminants

根据节瘤拟杆菌特有保守序列设计引物,利用聚合酶链式反应(PCR)建立了一种鉴别检测该菌的方法。对可能影响PCR检测结果的一系列因素进行了较详细的研究,选择了适宜PCR检测的快捷处理病样获得反应模板的方法,对PCR反应条件进行了优化,使对已知菌株培养物检测灵敏性最高可到30个菌/30μL反应体系;用广泛的相关菌株和菌群验证引物的特异性,证明设计的引物特异性强。将PCR方法用于临床病样的初步检测,部分病样PCR检测阳性。为用PCR方法检测节瘤拟杆菌引发的牛、羊、鹿腐蹄病奠定了基础。

Detection assay by PCR（Polymerase chain reaction） was constructed to D. nodosus, that can discriminate two group of Dichelobacter nodosus ： （1）Three primers were designed, based on the conserved region of aligned limA sequences of groups Ⅰ, Ⅱ of D. nodosus,of which forward primers were identical to group Ⅰ, Ⅱ, and reverse primers was specific to each group Ⅰ and group Ⅱ respectively; （2）The optimal PCR conditions were determined, in 30μl reaction volume the primers detected at least 30 cells of D. nodosus in crude lysates; （3）A serial of PCR assay verified the primers was specific toD. nodosus but not to the other bacterial and cells. The developed PCR technique were evaluated with directly lysate of samples from lesions footrot, the results found a few sample were positive in PCR test. From the results, which is suggested the developed PCR technique is adapted to identify D. nodosus and discriminate its serogroups, It will become a practical method to diagnosis of fo3trot in ruminants.