Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA.
BACKGROUND
Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR).


RESULTS
Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant(R) HBV-DNA Quantification Panel, Accrometrix Europe B.V.). The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67%) to the Procleix Ultrio discriminatory HBV test (dHBV) (70%) in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979).


CONCLUSIONS
We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.
Department of Hygiene Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece. dparask@med.uoa.gr
Dimitrios
D
Paraskevis
Apostolos
A
Beloukas
Catherine
C
Haida
Antigoni
A
Katsoulidou
Zisis
Z
Moschidis
Helen
H
Hatzitheodorou
Agoritsa
A
Varaklioti
Vana
V
Sypsa
Angelos
A
Hatzakis
D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral