Data Analysis and Graphical observations of real-time RT-PCR of Hepatitis C Virus to determine the range of qualitative assays and to differentiate it from quantitative assays

Hepatitis C Virus (HCV) load in plasma were often below the validated quantitative RT-PCR (RT-qPCR) calibration plot concentrations. In this report, data analysis of RT-PCR amplification plots and calibration plots were done to define the performance characteristics of the higher Cq range of qualitative RT-PCR and to differentiate it from RT-qPCR. This is the first report to establish the decision criteria for validation of qualitative real-time HCV RT-PCR assays. Patients reactive for anti-HCV antibody screening assays or exposed to HCV infection were selected for RT-PCR assay done with RNA isolated from plasma. Normalised fluorescence intensity (100%) and slope (1.65–1.50) of the sigmoid fluorescence amplification plots were of acceptable performance for validated quantitative calibrators (10,000–10 IU/µl), but were decreased for diluted calibrators and for patients’ sample with Cq greater than that of the 10 IU/µl validated calibrator (Cq > 33.2). Calibration plot of validated calibrators had slope, efficiency and sensitivity of -3.471, 94% and 36.404, respectively, but the diluted calibrators showed decreased performance of -2.305, 171% and 35.52, respectively. Log-transformed exponential amplification plots analysed by LinRegPCR software, showed acceptable performance at the validated calibrator concentrations and decreased performance with diluted calibrators and patients’ sample with Cq > 33.2. The four-parameter validated sigmoid fluorescence amplification plot of qualitative RT-PCR Cq range was from 33.2 to 39.9. The 10 IU/µl calibrator was the reference point to differentiate between qualitative and quantitative RT-PCR. A systematic decision-criteria had been developed for this purpose from the data analysis of graphical observations, four-parameter sigmoid curve fitting model, exponential amplifications of individual RT-PCR plots and calibration plots.