Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay.
BACKGROUND
Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV.


METHODS
The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay's capacity to detect genotypes prevalent among individuals attending this tertiary care hospital.


RESULTS
The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001).


CONCLUSIONS
This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.
Daniel
Hubert Darius J
HD
Department of Clinical Virology, Christian Medical College, Vellore, India.
Fletcher
John G
JG
Chandy
George M
GM
Abraham
Priya
P
D001782:Blood Donors; D004279:DNA, Viral / Q000097:blood*; D006515:Hepatitis B virus / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral