PCR Primers for The Detection of Propane and Butane-Oxidizing Microorganisms
PCR Primers for The Detection of Propane and Butane-Oxidizing Microorganisms
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DOI：
10.15368/theses.2011.12
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发表时间：
2011
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影响因子：
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通讯作者：
Brian Chan
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作者：
Brian Chan
在一个日益需要能源的世界中，我们满足未来更高的能源需求的能力已成为一个紧迫的问题。这些关切中最紧迫的是石油供应问题。许多专家都在鼓吹石油生产高峰即将到来以及随后产量下降的说法。为此，开发新的和先进的石油勘探方法已经变得几乎与寻找石油本身的来源一样重要。石油储层上方的土壤是各种烷烃氧化细菌群落的宿主，这些细菌可以利用储层排放的天然气作为碳和能量的来源。虽然甲烷可以来自非石油来源，但丙烷和丁烷的唯一天然来源是石油和天然气田。石油勘探者将给定土壤样品中丙烷和丁烷氧化细菌的增加用作近端石油储层的准确指示器。世纪以来，细胞计数和烃代谢率一直是用于确定烃氧化微生物存在的度量。这些方法需要数周才能完成。为了石油勘探的主要目的，我们开发了一套DNA引物，用于通过PCR扩增更快速地检测烃氧化微生物。每个引物的设计是基于来自七种生物的七个prmA和bmoX基因的核苷酸序列比对，其分别编码丙烷单加氧酶的大羟化酶亚基和丁烷单加氧酶的α羟化酶亚基。这些单加氧酶是负责引发丙烷和丁烷催化剂的酶。使用从已知的丁烷和丙烷氧化剂中提取的DNA作为阳性对照，并使用甲烷和甲苯氧化剂作为阴性对照，完成了使用该引物组的PCR优化。从丁烷氧化和丙烷氧化细菌的培养物和土壤样品中回收的PCR产物进行测序。从测序数据构建系统发育树以确认扩增的准确性。我们演示了使用PCR和琼脂糖凝胶电泳检测培养和复杂的微生物土壤群落中的烃氧化细菌。通过两个不同的实验阐明了检测限。潜在的进步途径包括通过选择性去除引物简并性来缩小特异性、使用额外的阳性和阴性对照以及使引物适应qPCR TaqMan测定。
In an increasingly energy-hungry world, our capacity to meet the heightened energy demands of the future has become a pressing matter. The most urgent of these concerns are tied to the accessibility of petroleum. Various experts have proselytized both the imminent arrival of peak oil production rates and the ensuing decline of those rates thereafter. And to that end, the development of novel and advanced oil exploration methodologies has become almost as important as finding the sources of oil themselves. The soils above petroleum reservoirs play host to various communities of alkane-oxidizing bacteria that can utilize the natural gas emitted by the reservoirs as a source of carbon and energy. While methane can originate from non-petroleum sources, the only natural sources of propane and butane are oil and gas fields. The increased presence of propane and butane-oxidizing bacteria in a given soil sample is used by oil prospectors as an accurate indicator of a proximal petroleum reservoirs. For over a century, cell counts and hydrocarbon metabolic rates have been the metrics used to determine the presence of hydrocarbon-oxidizing microbes. These methods require weeks to complete. Here, we have developed a set of DNA primers for a much more rapid detection of hydrocarbon-oxidizing microbes through PCR amplification-for the chief purpose of petroleum exploration. Each primer's design is based on a nucleotide sequence alignment of seven prmA and bmoX genes from seven organisms, which encode the large hydroxylase subunit of propane monooxygenase and V the alpha hydroxylase subunit of butane monooxygenase respectively. These monooxygenases are the enzymes responsible for the initiation of propane and butane catabolism. Optimization of PCR with this primer set was accomplished using DNA extracted from known butane and propane oxidizers as positive controls, and methane and toluene oxidizers as negative controls. PCR products recovered from cultures of butane-oxidizing and propane-oxidizing bacteria, and soil samples, were sequenced. Phylogenetic trees were constructed from the sequencing data to confirm the accuracy of amplification. We demonstrate the use of PCR and agarose gel electrophoresis to detect hydrocarbon-oxidizing bacteria in culture and in complex microbial soil communities. Detection limits were elucidated through two different experiments. Potential avenues of advancements include narrowing specificity by selectively removing primer degeneracies, the use of additional positive and negative controls and the adaptation of the primers to a qPCR TaqMan assay.