| ORIGINAL ARTICLE |
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| Year : 2011 | Volume
: 17
| Issue : 1 | Page : 17-21 |
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A latest and promising approach for prediction of viral load in hepatitis B virus infected patients
Naresh Yalamanchili1, Rahamathullah Syed1, Madhavi Chandra1, Vishnupriya Satti2, Ramachandra Rao1, Aejaz Habeeb Mohammed1, Khaja Mohammed Nanne1
1 Department of Gastroenterology, Centre for Liver Research and Diagnostics, Owaisi Hospital and Research Centre, Kanchanbagh, Hyderabad, India
2 Department of Genetics, Osmania University, Tarnaka, Hyderabad, India
Correspondence Address:
Naresh Yalamanchili
Centre for Liver Research and Diagnostics, Owaisi Hospital and Research Centre, Kanchanbagh, Hyderabad
India
 Source of Support: None, Conflict of Interest: None  | 2 |
DOI: 10.4103/0971-6866.83170
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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 10 2 to 10 8 . The calibration curve was linear in a range from 10 2 to 10 8 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. |
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