Kandpal JC, Kumar R, Kumar V, Singh PR
Abstract: Background A simple and reliable DNA extraction & quantification method of hepatitis B virus (HBV) is critical in developing a viral load measurement for HBV infection. Current commercially available plasma Hepatitis B Virus (HBV), DNA extraction & quantification methods are less sensitive and require optimization, which restrict wide adoption in clinical laboratories. This study offers a report on the quantitative viral load- artus HBV RG PCR assay by combining QIAamp DSP Plasma DNA extraction with Rotor gene Q-Real-time PCR (Rotor gene Q-PCR). Methods Plasma was separated by centrifugation after collection of whole peripheral blood, which was then stored at -200 C until process. DNA was extracted as per instruction & guidelines of manufacturer. The recovered eluted DNA was mixed into PCR master-mix. The Q-PCR assay performance, including linearity, diagnostic accuracy and reliability were determined. Viral load compared of the no viral load, low viral titer & high viral titer from the clinical samples for evaluation Results The average % yield for HBV DNA standards used was within in the range (SD 0.04 to 0.13, CV % 0.97 to 2.22). The coefficient of variation (CV) of HBV quantification for low and high titer samples was 2.23 % and 2.37 %, respectively. The real-time assay linearity was demonstrated with a slope of -3.03 to -3.50 and R2 values of 0.98 to 0.99. Accuracy and reliability of the Rotor gene Q-PCR assay was confirmed with the reference panel, and there was a strong correlation between the two results (R2 = 0.99, < 0.01) & found no major difference in observed viral load. Conclusions The Rotorgene Q-PCR performance with artus-DSP assay is reliable and wide linear range quantification method and can be used for sensitive detection of plasma HBV with low nucleic acids content.
Keywords: Hepatitis B Virus, Quantitative Standards, DNA amplification, Taqman Technology