Real time TaqMan PCR detection and quantitation of HBV genotypes A-G with the use of an internal quantitation standard.
BACKGROUND
Diagnostic assays for the accurate quantitation of hepatitis B virus (HBV) DNA levels from patients undergoing antiviral therapy are useful for monitoring and tailoring therapy. Such assays should give accurate results with all HBV genotypes, including a seventh genotype of hepatitis B virus, genotype G, that has recently been identified in specimens from HBV-positive patients in the United States and Europe.


OBJECTIVE
To characterize the performance characteristics of a quantitative real time TaqMan PCR assay, the High Pure System Viral Nucleic Acid/COBAS TaqMan HBV Test, for the detection and quantitation of HBV genotypes A-G from patient plasma and serum. This test was evaluated for limit of detection, dynamic range, reproducibility, accuracy, and genotype inclusivity.


METHODS
Primers and TaqMan probes specific for HBV and an internal quantitation standard (QS) were designed and tested using a set of plasmid DNAs representing various genotyped specimens. In addition, sensitivity, dynamic range, precision, and correlation with the COBAS AMPLICOR HBV MONITOR Test were evaluated using HBV dilution panels and patient specimens.


CONCLUSIONS
A real time TaqMan assay was developed that detects and quantifies DNA from genotypes A-G equivalently. The assay has a limit of detection of <50 copies/ml with plasma and serum, a dynamic range up to 10(9) copies/ml, and good precision and accuracy. Titers obtained with this method without dilutions show good correlation across the dynamic range with titers obtained with the COBAS AMPLICOR HBV MONITOR Test.
Weiss
J
J
Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, CA 94588, USA.
Wu
H
H
Farrenkopf
B
B
Schultz
T
T
Song
G
G
Shah
S
S
Siegel
J
J
D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood; D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load
D017931:DNA Primers; D004279:DNA, Viral