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

开发具有成本效益的实时 PCR 检测方法:用于检测巴西亚马逊西部地区的各种 HBV DNA 浓度

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作者:Alcionede de Oliveira dos Santos, Luan Felipo Botelho Souza, Lourdes Maria Borzacov, Juan Miguel Villalobos-Salcedo, Deusilene Souza Vieira

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

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.

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 × 10&sup9; 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.

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