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

摘要

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? 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.