Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western Amazon region of Brazil.
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.
Fundação Oswaldo Cruz, Rondônia-FIOCRUZ, Porto Velho, Rondonia, Brazil. alcione.m@hotmail.com.
Alcionede de Oliveira
Ade O
dos Santos
Luan Felipo Botelho
LF
Souza
Lourdes Maria
LM
Borzacov
Juan Miguel
JM
Villalobos-Salcedo
Deusilene Souza
DS
Vieira
D001938:Brazil; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*