油气田土壤甲烷氧化菌实时荧光定量PCR检测技术的建立与应用

Development and application of a fluorescent quantitative real-time PCR technique for detection of methane-oxidizing bacteria in oil and gas field soil

【目的】建立快速检测甲烷氧化菌含量的SYBR GreenⅠ实时荧光定量PCR技术,用于油气微生物勘探。【方法】以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测。【结果】该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976%,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3%,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性。利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值。【结论】为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考。

[Objective] We developed a rapid SYBR Green I fluorescent quantitative real-time PCR technique for detection of methane-oxidizing bacteria for microbial prospection of oil and gas. [Methods] The recombinant plasmid containing pmoA gene was used as standards to optimize theexperimental conditions and generate a standard curve. The sensitivity, reproducibility and specificity of the technique were evaluated, and practical soil samples were detected using the technique. [Results] The correlation coefficient （R^2） of the standard curve was 0.999 9 and the amplification efficiency was99.976%. The detection range was from 3.897×10^1 to 3.897×10^9 copies/μL and the sensitivity was about 40 copies/μL. All variable coefficients of CT values in the reproducibility test were better than 3%. In addition, no amplification was observed in the templates of 12 non-methane-oxidizingbacteria. The technique showed good sensitivity, specificity and reproducibility. Soil samples collected in the gas field, the oil field and the non-oil and gas field were detected using the technique, and the gas field had high anomalies of methane-oxidizing bacteria. [Conclusion] The fluorescencequantitative real-time PCR technique developed here could rapidly and exactly detect methane-oxidizing bacteria for oil and gas exploration. At the same time, this technique could contribute to detection of other indicator bacteria in oil and gas field soil.