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

作者：dos Santos Alcione de Oliveira^*; Botelho Souza Luan Felipo; Borzacov Lourdes Maria; Villalobos Salcedo Juan Miguel; Vieira Deusilene Souza

来源：Virology Journal, 2014, 11(1): 16.

DOI：10.1186/1743-422X-11-16

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 (R) HBV-DNA Quantification Panel, Acrometrix Europe B.V.).
Results: From our in-house plasmid we prepared serial dilutions ranging from 2 x 10(3) - 2 x 10(9) copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r(2) = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 mu l. We found a strong correlation between the two methods (r(2) = 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.

• 出版日期2014-1-28