Abstract：
BACKGROUND: Quantitative polymerase chain reaction (qPCR) is a molecular method used in laboratories for detection and quantification of DNA nucleotides. Standard curves and reference values are usually combined into the PCR amplification process to calculate initial amount of target DNA molecule in a testing sample. This study is a retrospective review to examine the performance of standard curves in a qPCR-based assay for the screening of a genetic disorder in laboratories.
METHODS: Three different scenarios were designed to replay the historical data generated from over 3,000 qPCR assays to evaluate how omission of standard curves would affect the overall screening results.
RESULTS: The outcomes of all the scenarios concluded that including standard curves in qPCR assays had decreased the screening specificity and accuracy, resulting in more false positives and additional retests. This was particularly true in case the initial DNA molecules were at relatively low copy numbers.
CONCLUSIONS: It strongly suggests that the implantation of qPCR assays in diagnostic procedures should be frequently retested and reviewed. More importantly, the strategies of retrospective review and scenario analysis presented here will provide a good framework for assay validation and periodic quality assurance in laboratory.
METHODS: Three different scenarios were designed to replay the historical data generated from over 3,000 qPCR assays to evaluate how omission of standard curves would affect the overall screening results.
RESULTS: The outcomes of all the scenarios concluded that including standard curves in qPCR assays had decreased the screening specificity and accuracy, resulting in more false positives and additional retests. This was particularly true in case the initial DNA molecules were at relatively low copy numbers.
CONCLUSIONS: It strongly suggests that the implantation of qPCR assays in diagnostic procedures should be frequently retested and reviewed. More importantly, the strategies of retrospective review and scenario analysis presented here will provide a good framework for assay validation and periodic quality assurance in laboratory.
摘要：
背景：定量聚合酶链反应（qPCR）是实验室中用于检测和定量DNA核苷酸的分子方法。通常将标准曲线和参考值结合到PCR扩增过程中以计算测试样品中的靶DNA分子的初始量。这项研究是一项回顾性审查，以检查基于qPCR的测定中标准曲线的性能，用于在实验室中筛查遗传疾病。
方法：设计了三种不同的方案来重放从超过3,000个qPCR测定产生的历史数据，以评估标准曲线的遗漏将如何影响整体筛选结果。
结果：所有方案的结果都得出结论，在qPCR分析中包括标准曲线会降低筛选特异性和准确性，导致更多的假阳性和额外的重新测试。在初始DNA分子处于相对低的拷贝数的情况下尤其如此。
结论：这强烈表明，在诊断程序中植入qPCR测定应经常重新测试和审查。更重要的是,本文提出的回顾性回顾和情景分析策略将为实验室的分析验证和定期质量保证提供良好的框架.
方法：设计了三种不同的方案来重放从超过3,000个qPCR测定产生的历史数据，以评估标准曲线的遗漏将如何影响整体筛选结果。
结果：所有方案的结果都得出结论，在qPCR分析中包括标准曲线会降低筛选特异性和准确性，导致更多的假阳性和额外的重新测试。在初始DNA分子处于相对低的拷贝数的情况下尤其如此。
结论：这强烈表明，在诊断程序中植入qPCR测定应经常重新测试和审查。更重要的是,本文提出的回顾性回顾和情景分析策略将为实验室的分析验证和定期质量保证提供良好的框架.
