| 沉积物中氨氧化细菌amoA基因荧光定量PCR检测方法的改进 |
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| 引用本文： | 路兴岚,甄毓,米铁柱,于志刚.沉积物中氨氧化细菌amoA基因荧光定量PCR检测方法的改进[J].中国海洋大学学报(自然科学版),2012(Z1):176-181. |
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| 作者姓名： | 路兴岚 甄毓 米铁柱 于志刚 |
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| 作者单位： | 中国海洋大学环境科学与工程学院;中国海洋大学海洋环境与生态教育部重点实验室;中国海洋大学化学化工学院;中国海洋大学教育部海洋化学理论与工程技术重点实验室 |
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| 基金项目： | 国家自然科学基金重大国际(地区)合作项目(40920164004);国家自然科学基金项目(40976044);海洋公益性行业科研专项项目(201105014)资助 |
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| 摘 要： | 聚合酶链式反应(Polymerase chain reaction,PCR)较容易受到某些抑制成分的影响,尤其是在对复杂环境样品进行的荧光定量PCR反应中,定量结果的准确性更易受到影响。为减轻这种抑制作用,以采自长江口邻近海域沉积物的DNA为研究对象,采用稀释模板法和添加BSA法对荧光定量PCR反应进行了优化。结果表明2种方法都有减轻抑制的作用,但若考虑方法的准确性和检测的便捷性,则以添加BSA的方法更为优越,最适添加浓度为0.1～0.5μg/μL。这一检测方法的改进,为研究沉积物中微生物在硝化过程中的重要作用提供了可靠的技术途径。
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| 关 键 词： | 荧光定量PCR 牛血清蛋白 沉积物 amoA基因 |
Improvements of Quantitative Real-Time PCR Assay for amoA Gene of Aerobic Ammonium Oxidizing Bacteria in Sediments |
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| LU Xing-Lan,ZHEN Yu,MI Tie-Zhu,YU Zhi-Gang.Improvements of Quantitative Real-Time PCR Assay for amoA Gene of Aerobic Ammonium Oxidizing Bacteria in Sediments[J].Periodical of Ocean University of China,2012(Z1):176-181. |
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| Authors: | LU Xing-Lan ZHEN Yu MI Tie-Zhu YU Zhi-Gang |
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| Institution: | 3,4(1.College of Environmental Science and Engineering;2.Key laboratory of Marine Environment and Ecology,Ministry of Education;3.College of Chemistry and Chemical Engineering;4.Key Laboratory of Marine Chemistry Theory and Technology,Ministry of Education,College of Chemistry and Chemical Engineering,Ocean University of China,Qingdao 266100,China) |
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| Abstract: | Polymerase chain reaction(PCR) is easily and negatively affected by the inhibitors especially when complex environmental samples are analyzed with quantitative real-time PCR(qRT-PCR),which leads to lower quantitation accuracy.Two methods of diluting template and adding bovine serum albumin(BSA) were used to optimize the qRT-PCR assay in order to reduce inhibition.The environmental DNA template was extracted from the sediments of adjacent waters of Changjiang Estuary.The result showed that the goal of reducing inhibition was achieved with the two methods,but the latter was better when the accuracy and convenience were taken into account.The optimum concentration of BSA was 0.1~0.5 μg/μL.The improved quantitative real-time PCR assay will provid a reliable way for studying the role of microorganisms in the nitrification process in sediments. |
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| Keywords: | quantitative real-time PCR bovine serum albumin sediment amoA gene |
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