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

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 {"type":"entrez-nucleotide","attrs":{"text":"EU684022","term_id":"189176131","term_text":"EU684022"}}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.