Determination of Real‐Time Polymerase Chain Reaction Uncertainty of Measurement Using Replicate Analysis and a Graphical User Interface with Fieller’s Theorem

Abstract

BACKGROUND:Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods.OBJECTIVE:To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays.METHODS:Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller’s theorem) and PCR efficiency.RESULTS:The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests.DISCUSSION:A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2

“…Further, based on indigenous F-phage analysis Hata and Honda (2020) reported a high efficiency of PEG method in Japanese wastewater, yet an evaluation of sample concentration efficiency, using the whole process control (WPC) together with MPC is recommended ( Haramoto et al, 2018 ). We recommend longer monitoring with several replicated analyses to evaluate the correlation as well as uncertainties involving RT-PCR ( Stuart et al, 2014 ) and then replace the semi-quantitative method employed in this study with precise copy calculations using suitable methods.…”