Department of Global Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, 1440 Canal Street, Suite 2100, New Orleans, LA, 70112, USA.
U.S. Environmental Protection Agency, National Risk Management Research Laboratory, 26 W. M.L. King Dr, Cincinnati, OH, 45268, USA.
Water Res. 2019 Jun 1;156:465-474. doi: 10.1016/j.watres.2019.03.014. Epub 2019 Mar 16.
There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials.
人们对应用快速定量聚合酶链反应（qPCR）方法来监测休闲淡水水质中的粪便指示菌大肠杆菌（E. coli）感兴趣。在这项研究中，我们确定了 21 个实验室在满足拟议的标准化数据质量验收（QA）标准方面的表现，以及这些实验室在分析美国环保署（EPA）为 E. coli 开发的草案 qPCR 方法的 18 个共享地表水样本时目标基因拷贝估计值的变异性。参与的实验室范围从具有更广泛 qPCR 经验的学术和政府实验室到“新”的水质和公共卫生实验室，在大多数情况下，这些实验室相对缺乏先前的经验。在总共 376 个测试样本分析中，有 24%的分析未能达到该方法的 QA 标准。在这些失败中，有 39%来自两个“新”实验室。可能导致 QA 失败的因素包括在推荐的参考和控制材料的储存和制备程序中存在偏差。研究还发现，与之前 EPA 的 qPCR 方法中使用的 delta-delta Ct（ΔΔCt）校准模型相比，主标准曲线校准模型更能降低目标基因拷贝估计值的对数总体变异性。然而，两种模型的平均估计值之间没有显著差异，并且在任何情况下，实验室之间的变异性都是导致方法总体变异性的最大因素。研究结果表明，多个实验室采用这种或其他具有类似数据质量验收标准的 qPCR 水质监测方法具有技术可行性，但建议即使对于一些更普遍有经验的 qPCR 实验室，也可能需要额外的实践和/或帮助。应特别注意提供和遵循关于参考和控制材料的制备、储存和处理的明确指导。
