Virus Quantitative Polymerase Chain Reaction Research Articles

An analysis of the standard curve parameters of cytomegalovirus, BK virus and hepatitis B virus quantitative polymerase chain reaction from a clinical virology laboratory in eastern India

PurposeQuantitative real-time polymerase chain reactions (qPCRs) are important for accurate detection of nucleic acid target including that for viral load determination. Assessment of the quality of a PCR run is essential for quality control, diagnostics and research. In order to reduce subjectivity qPCR standard curves are accompanied with parametric values for slope, Y- intercept, correlation coefficient (R2) and PCR efficiency. In this study the performance of three qPCR assays-cytomegalovirus, hepatitis B virus and BK virus-with respect to standard curve parameters-slope, Y intercept, R2 and efficiency were examined. MethodsUsing ideal values (Slope (minus 3.32); Y intercept ​= ​the number of PCR cycles; R2 ​= ​1 and efficiency ​= ​100%) we estimated the intra-assay variability (range) and deviation from ideal parameters (Δ). We also calculated the standard deviation (SD) and coefficient of variation (CV) for each of these parameters. We have evaluated the quality of each of the three viral load assays (CMV, HBV, BKV) using these statistical approach. ResultsWe found lab developed tests (CMV) to have least deviation from ideal Y intercept (limit of detection); however, commercial kit based assays had better linearity (scatter plot correlation between amplification factor and PCR efficiency). Using a scatter plot for the three assays we found the correlation with calculated amplification factor and PCR efficiency was most linear in case of BKV (0.9974), closely followed by the HBV assay (R2 ​= ​0.9968). Although the CMV quantitative standards were least linear (0.868), the CV (coefficient of variation) was also the least in case of the CMV assay. ConclusionThe study highlights an objective way of assessing qPCR assay quality and demonstrates a method to compare assays, validate tests and perform quality control.

Cross-validation Studies of a Novel Low-cost Hepatitis B Virus Quantitative Polymerase Chain Reaction System.

AimThis research focused on the results of the cross-validation program related with the performance of a Cuban novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay for hepatitis B virus (HBV) quantification developed by the Therapeutic Vaccine against Hepatitis B Department, Vaccines Division, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.Materials and methodsDilution series with the plasmid standard at concentrations of 900,000 to 0.09 copies/reaction (c/r) were made for each PCR instrument. The mean cycles threshold (Ct) values and PCR efficiency were compared among the cyclers. Hepatitis B virus-positive serum samples were used for the calculation of reproducibility of the HBV assay. Biotecon Diagnostics (BCD) also ordered the oligo sequences from a second supplier and compared the PCR performance to those provided from the CIGB.ResultsAll PCR cyclers were able to detect concentrations up to 0.09 c/r. However, below the concentration of 9 c/r, the variation of results increased within and between the cyclers. The PCR efficiency showed satisfying results. The overall coefficient of variation (CV) cycler values were 1.29 and 0.91% for M6 and M19 respectively. No significance was observed between the different primer suppliers.ConclusionThe HBV assay was performed with a good concordance between the five real-time instruments from different suppliers. The HBV assay was also performed with a high reproducibility for samples with a high and a low viral load. The HBV assay is robust against different primer suppliers.How to cite this article: Aguiar J, Silva JA, García G, Guillén G, Aguilar JC. Cross-validation Studies of a Novel Low-cost Hepatitis B Virus Quantitative Polymerase Chain Reaction System. Euroasian J Hepato-Gastroenterol 2018;8(1):38-41.