应用竞争PCR方法检测亚硝化细菌的研究  

The Research of Detection Method to Nitrite Bacteria by Competitive PCR

作　　者：张艳[1] 李秋芬[1] 王晓熙[1,2] 

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛266071 [2]中国海洋大学生命科学与技术学院,青岛266003

出　　处：《生物技术通报》2009年第S1期394-397,409,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30200209);农业部海洋生物资源可持续利用重点开放实验室院士基金特别资助项目

摘　　要：选用竞争PCR技术建立了亚硝化细菌的快速定量检测方法。实验以实验室分离、鉴定的一株亚硝化细菌提取的DNA为模板,设计特异性引物,制备特异性内标模板,进行竞争PCR实验;同时采用传统的平板计数法和吖啶橙荧光显微镜计数法两种方法进行细菌计数,与竞争PCR结果比较分析,建立了样品细菌数量与内标模板量的线性方程,并将其应用于海水养殖环境中亚硝化细菌的检测中。该方法具有简化检测过程、缩短细菌检测时间、无须昂贵仪器设备等优点,并克服有些环境微生物难以培养的困难,为将来有益菌在应用中实际存活和繁殖情况的监测提供了有力的技术支撑。Competitive polymerase chain reaction assay(cPCR) was used to establish quickly detecting method of nitrite bacteria in this paper.This experiment included taking count of bacteria sample,extracting DNA,designing differential primers and internal control template.Two methods were applied to count the barteria:Plate count for bacterial colonies and acridine orange fluorescence staining microscopy count;Releasing DNA adopted storing at -20℃and boiling,the results indicated that the method of store at -20℃was better than boiling;Based on PCR assay,a 613bp fragment was amplified and subcloned into pMD18-T vector,the 265bp truncated and homologous fragment was the internal control template.By the same pair of primers,one series of dilutions of internal control templates was amplified together with the same volume of target bacterial DNA in the same PCR reaction,analyzing the competitive PCR and bacteria count results,a linearity equation was established.The result indicated that there was distinctly competitive linear relationship between original target DNA and internal control DNA,by which,nitrite bacteria of environment was detected.Comparing with the traditional methods,this method has many advantages,such as:saving time、avoiding incubating bacteria etc.

关 键 词：竞争PCR 亚硝化细菌 定量检测 内标模板 

分 类 号：Q93[生物学—微生物学]