A latest and promising approach for prediction of viral load in hepatitis B virus infected patients.
Loading...
Date
2011-01
Journal Title
Journal ISSN
Volume Title
Publisher
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 {"type":"entrez-nucleotide","attrs":{"text":"EU684022","term_id":"189176131"}}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.
Description
Keywords
Hepatitis B virus, quantification, real-time Polymerase Chain Reaction, TaqMan chemistry 19p 13.3, genetic markers
Citation
Yalamanchili Naresh, Syed Rahamathullah, Chandra Madhavi, Satti Vishnupriya, Rao Ramachandra, Mohammed Aejaz Habeeb, Nanne Khaja Mohammed. A latest and promising approach for prediction of viral load in hepatitis B virus infected patients. Indian Journal of Human Genetics. 2011 Jan; 17(1): 17-21.