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Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

Alcione de Oliveira dos Santos123*, Luan Felipo Botelho Souza123, Lourdes Maria Borzacov2, Juan Miguel Villalobos-Salcedo123 and Deusilene Souza Vieira123

Author Affiliations

1 Fundação Oswaldo Cruz, Rondônia-FIOCRUZ, Porto Velho, Rondonia, Brazil

2 Centro de Pesquisa em Medicina Tropical de Rondônia-CEPEM, Porto Velho, Rondonia, Brazil

3 Universidade Federal de Rondonia-UNIR, Porto Velho, Rondonia, Brazil

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Virology Journal 2014, 11:16  doi:10.1186/1743-422X-11-16

Published: 28 January 2014

Abstract

Background

Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT).

Methods

A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.).

Results

From our in-house plasmid we prepared serial dilutions ranging from 2 × 103 – 2 × 109 copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r2 = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r2 = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL.

Conclusions

Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions.