CAJ | 학술논문

建立了SYBR Green I实时荧光定量聚合酶链式反应( Real Time-qPCR)检测油田污染土壤中烷烃降解基因AlkB和萘降解基因Nah的方法。比对相关降解石油菌株的GenBank序列,设计合成针对烷烃和萘降解基因扩增引物AlkBf/AlkBr和Nahf/Nahr。将纯化的常规PCR胶回收产物与pEASY-T1载体连接,转化到感受态细胞培养。提取并梯度稀释阳性克隆质粒,构建Real Time-qPCR标准测定曲线。25μL扩增体系最佳反应条件：前后引物终浓度为0.2μmol/L,12.5μL 2×TransStart Top Green qPCR SuperMix,AlkB和Nah基因最适退火温度分别为50℃和57℃。 Real Time-qPCR技术显示出很高的灵敏性和重复性,比传统PCR技术灵敏度高100倍。对采集于某油田3个功能区的14土壤样品中AlkB定量检测显示,石油污染严重的采油区含有最高的AlkB拷贝数,污染较轻的生活区AlkB拷贝数最少；Nah基因分布均匀。
건립료SYBR Green I실시형광정량취합매련식반응( Real Time-qPCR)검측유전오염토양중완경강해기인AlkB화내강해기인Nah적방법。비대상관강해석유균주적GenBank서렬,설계합성침대완경화내강해기인확증인물AlkBf/AlkBr화Nahf/Nahr。장순화적상규PCR효회수산물여pEASY-T1재체련접,전화도감수태세포배양。제취병제도희석양성극륭질립,구건Real Time-qPCR표준측정곡선。25μL확증체계최가반응조건：전후인물종농도위0.2μmol/L,12.5μL 2×TransStart Top Green qPCR SuperMix,AlkB화Nah기인최괄퇴화온도분별위50℃화57℃。 Real Time-qPCR기술현시출흔고적령민성화중복성,비전통PCR기술령민도고100배。대채집우모유전3개공능구적14토양양품중AlkB정량검측현시,석유오염엄중적채유구함유최고적AlkB고패수,오염교경적생활구AlkB고패수최소；Nah기인분포균균。
SYBR Green I Real Time-qPCR method was developed to quantify the numbers of copyies of AlkB ( alkanes degradation gene) and Nah ( naphthalene dioxygenase degradation gene) functional degradation gene corresponding to alkanes and aromatic hydrocarbons degradation. Two pairs of primers AlkBf/AlkBr and Nahf/Nahr were designed for AlkB and Nah amplification respectively, according to the nucleotide sequences of related degradation microorganisms published in GenBank. The purified recovery products of traditional PCR were combined with pEASY-T1 vectors and transformed in competent cells to amplify. The recombinant plasmids were extracted and used as positive templates to create standard curve through gradient dilution. The conditions for the real time PCR were as the follows: the final concentration of forward and reverse primers were 0. 2 μmol/L, 2×TransStart Top Green qPCR SuperMix, and the annealing temperatures of AlkB and Nah PCR were 50℃ and 57℃, respectively. The method showed a sensitivity of 100 times higher than that of the traditional PCR method and good repeatability. The numbers of copies of AlkB in three functional regions of an oilfield indicated that oil producing zone with serious oil pollution had the highest AlkB copy numbers, and residential zone with lighter oil pollution had the lowest AlkB copy numbers. Nah degradation gene distribution was more uniform.