A real-time polymerase chain reaction method for monitoring anaerobic, hydrotarbon-degrading bacteria based on a catabolic gene
A real-time polymerase chain reaction method for monitoring anaerobic, hydrotarbon-degrading bacteria based on a catabolic gene
复制标题
DOI：
10.1021/es025556w
复制
发表时间：
2002-09-15
影响因子：
11.4
通讯作者：
Alvarez, PJJ
中科院分区：
文献类型：
--
作者：
Beller, HR;Kane, SR;Alvarez, PJJ
我们已经开发了一种实时聚合酶链反应（PCR）方法，可以量化的烃降解细菌在沉积物样品的基础上的分解代谢基因与厌氧甲苯和二甲苯降解的第一步。靶基因bssA编码苄基琥珀酸合酶的α-亚基。用于实时PCR的引物-探针集基于来自四种细菌菌株的bssA的共有区域;在本研究期间确定了其中两种菌株的bssA序列。该方法灵敏度高（检出限约为0.05 μ g/ml）。5个基因拷贝），并且具有>7个数量级的线性范围。我们使用的方法来调查如何，乙醇汽油泄漏的地下储罐释放可能会影响土著甲苯降解细菌。接种从四个不同的网站与含水层沉积物的缩影厌氧培养BTEX（苯，甲苯，二甲苯）和硝酸盐在乙醇的存在和不存在下。总体而言，种群趋势与观察到的甲苯降解活性一致：甲苯降解最快的微宇宙也最大。bssA拷贝数。在甲苯降解最快的微观环境中，bssA拷贝数在孵育的前4天增加了100至1000倍，在此期间，大部分甲苯。已经被消耗了。这些结果得到了未扩增DNA的狭缝印迹分析以及推定的bssA扩增子的克隆和测序的支持，这证实了实时PCR方法对bssA的特异性。使用伴侣实时PCR方法估计总的真细菌种群（基于16 S rDNA）表明，在某些情况下，乙醇不成比例地支持不含bssA的细菌的生长。bssA的实时PCR方法可以是一种。监测受燃料污染的地下水中BTEX自然衰减的强大工具。据我们所知，这是第一个报道的分子方法，目标厌氧，烃降解细菌的基础上分解代谢基因。
We have developed a real-time polymerase chain reaction,(PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation. The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase. The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of >7 orders of magnitude. We used the method to investigate how, gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria. Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol. Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest. numbers of bssA copies. In the microcosms with the most rapid toluene degradation, numbers,of bssA copies increased 100- to 1000-fold over the, first 4 days of incubation, during which time most of the toluene. had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA. Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases ethanol disproportionately supported the growth of bacteria that did not contain bssA. The real-time, PCR method for bssA could be a. powerful tool for monitored natural, attenuation of BTEX in fuel-contaminated groundwater. To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.