Raggi Claudia Casini, Verderio Paolo, Pazzagli Mario, Marubini Ettore, Simi Lisa, Pinzani Pamela, Paradiso Angelo, Orlando Claudio
Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Italy.
Clin Chem Lab Med. 2005;43(5):542-8. doi: 10.1515/CCLM.2005.094.
Quantitative real-time PCR techniques are increasingly being used for the measurement of nucleic acids in research applications as well as in the clinical laboratory. It is therefore important that external quality control programs (EQA) are implemented for the evaluation of the analytical aspects common to molecular tests based on quantitative PCR. The aim of this study was the development of an Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Mantrade mark probes to compare the analytical performance of 42 laboratories. Participants were provided with a set of reagents (cDNA for reference curve preparation, primers-probe mix and three unknown samples) and requested to perform a conventional assay using the master mix employed in their laboratories. The quantitative results in unknown samples were analyzed. The results of our study showed clear heterogeneity in performance. Two of the 42 laboratories provided results indicating contamination during the experiment, whereas six did not provide values for at least one of the six standard points. Only 12 laboratories gave results that were both precise and accurate for all the samples tested. Regarding imprecision, 17 laboratories appeared to deviate in at least one result, whereas inaccuracy showed an inverse dose-dependent trend. Finally, 12 laboratories were not able to measure the sample with the lowest concentration. Ten of these laboratories were equipped with the same instruments. The results of this first round of analytical EQA of real-time PCR-based methods seem to indicate high variability among laboratories carrying out the same experimental protocol. These findings could have implications for any assay based on this type of technique. This survey demonstrates the importance of experimental EQAs of methodological proficiency testing. Our approach has proved useful for comparing the analytical aspects shared by all diagnostic laboratories applying quantitative assays for the measurement of nucleic acids based on the use of Taq-Mantrade mark probes and real-time platforms.
定量实时聚合酶链反应（PCR）技术在研究应用以及临床实验室中越来越多地用于核酸检测。因此，实施外部质量控制程序（EQA）对于评估基于定量PCR的分子检测共有的分析方面非常重要。本研究的目的是开发一个意大利的基于Taq-Man™探针实时PCR定量检测的外部质量控制程序，以比较42个实验室的分析性能。为参与者提供了一组试剂（用于制备标准曲线的cDNA、引物-探针混合物和三个未知样品），并要求他们使用实验室中使用的预混液进行常规检测。分析未知样品中的定量结果。我们的研究结果显示性能存在明显异质性。42个实验室中有两个提供的结果表明实验过程中存在污染，而六个实验室至少有一个六个标准点未提供数值。只有12个实验室对所有测试样品给出了精确且准确的结果。关于不精密度，17个实验室似乎至少有一个结果出现偏差，而不准确则呈现出相反的剂量依赖性趋势。最后，12个实验室无法检测最低浓度的样品。其中十个实验室配备了相同的仪器。基于实时PCR方法的第一轮分析EQA结果似乎表明，执行相同实验方案的实验室之间存在高度变异性。这些发现可能对基于此类技术的任何检测都有影响。本次调查证明了方法学能力验证实验EQA的重要性。我们的方法已被证明有助于比较所有应用基于Taq-Man™探针和实时平台的定量检测来测量核酸的诊断实验室共有的分析方面。
