A latest and promising approach for prediction of viral load in hepatitis B virus infected patients

Abstract

INTRODUCTION:Designing a rapid, reliable and sensitive assay for detection of hepatitis B virus (HBV) variants by real-time PCR is challenging at best. A recent approach for quantifying the viral load using a sensitive fluorescent principle was brushed in this study.MATERIALS AND METHODS: A total of 250 samples were collected from the outpatient unit, CLRD. Complete Human HBVDNA sequences (n = 944) were selected from the National Centre for Biotechnology Information (NCBI), primers and probes were designed and synthesized from the core, surface, and x region. Real-time based quantification was carried out using a standard kit and in-house generated standards and RT-PCR protocols.RESULTS AND DISCUSSION:The standard calibration curve was generated by using serial dilution 102 to 108. The calibration curve was linear in a range from 102 to 108 copies/ml, with an R2 value of 0.999. Reproducibility as measured by dual testing of triplicates of serum samples was acceptable, with coefficients of variation at 6.5%, 7.5%, and 10.5%. Our results showed that amplification performance was good in the case of the x-region-based design (98%). Out of 100 negative samples screened by enzyme linked immunosorbent assay and the standard RT-PCR kit, one sample was detected as positive with the in-house developed RT-PCR assay, the positivity of the sample was confirmed by sequencing the amplified product, NCBI accession EU684022.CONCLUSION:This assay is reproducible showing limited inter- and intra-assay variability. We demonstrate that the results of our assay correlated well with the standard kit for the HBV viral load monitor.

Mini review: Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1

“…Their method displayed linearity over a range of 2 Â 10 3 to 2 Â 10 9 copies/ml and a lower limit of detection of 2000 IU/ml. Yalamanchili et al 24 designed a rapid, reliable, and sensitive assay for the detection of HBV by RT-PCR. The calibration curve was linear over a range of 1 Â 10 2 to 1 Â 10 8 copies/ml, with an R 2 value of 0.999.…”