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Interlaboratory Comparison of Real-Time PCR Protocols for Quantification of General Fecal Indicator Bacteria

机译:实时PCR协议实验室间比较,用于定量一般粪便指示剂细菌

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摘要

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Enterola and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.
机译:在美国,正在考虑将定量实时PCR(qPCR)技术用于快速鉴定环境水中的粪便细菌。从研究工具到标准化协议的过渡,需要有关跨实验室qPCR方法学的可重复性和变异来源的信息。这项研究分别检查了使用Enterola和GenBac3 qPCR方法从标准,加标和环境DNA来源中测量肠球菌和拟杆菌浓度的实验室间差异。比较是基于来自八个不同研究机构的数据。特别注意的是DNA分离步骤的影响以及单纯形和多重扩增方法对实验室间变异性的影响。结果表明,除非环境样品中含有能抑制qPCR扩增的物质,否则粗裂解物足以用于DNA分离。在单纯和多重扩增方法之间没有观察到明显的差异。总体而言,无论qPCR方案如何,实验室间的变异性水平均保持较低(变异系数<10%)。

著录项

  • 来源
    《Environmental Science & Technology》 |2012年第2期|p.945-953|共9页
  • 作者单位

    U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio, United States;

    University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, North Carolina, United States;

    University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, North Carolina, United States;

    University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, North Carolina, United States;

    U.S. Geological Survey, Columbus, Ohio, United States;

    U.S. Geological Survey, Columbus, Ohio, United States;

    City of Racine Health Department, Racine, Wisconsin, United States;

    City of Racine Health Department, Racine, Wisconsin, United States;

    National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratories, Ocean Chemistry Division, Miami, Florida, United States;

    National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratories, Ocean Chemistry Division, Miami, Florida, United States;

    Southern California Coastal Water Research Project, Costa Mesa, California, United States;

    Southern California Coastal Water Research Project, Costa Mesa, California, United States;

    Southern California Coastal Water Research Project, Costa Mesa, California, United States;

    Department of Biology, University of South Florida, Tampa, Florida, United States;

    Department of Biology, University of South Florida, Tampa, Florida, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, Ohio, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, Ohio, United States;

    U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, Ohio, United States;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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