Retrospective Review and Scenario Analysis for qPCR Quality Assurance.

Abstract

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. 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. 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. 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.