title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: London Health Sciences Centre and Western University, London, Ontario.
authors.forename: James Ian
authors.initials: JI
authors.lastname: Stuart
authors.affiliation: London Health Sciences Centre and Western University, London, Ontario.
authors.forename: Johan
authors.initials: J
authors.lastname: Delport
authors.affiliation: London Health Sciences Centre and Western University, London, Ontario.
authors.forename: Robert
authors.initials: R
authors.lastname: Lannigan
authors.affiliation: London Health Sciences Centre and Western University, London, Ontario.
authors.forename: George
authors.initials: G
authors.lastname: Zahariadis
keywords: BK virus; CMV; EBV; Graphical user interface; Quantitative real-time PCR; Uncertainty of measurement