Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes.
BACKGROUND
TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants.


METHODS
TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method.


RESULTS
The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results.


CONCLUSIONS
We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.
Hua
Rui
R
Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan.
Tanaka
Yasuo
Y
Fukai
Kenichi
K
Tada
Motohisa
M
Seto
Motoko
M
Asaoka
Yoshinari
Y
Ohta
Miki
M
Goto
Tadashi
T
Kanai
Fumihiko
F
Kato
Naoya
N
Yoshida
Haruhiko
H
Kawabe
Takao
T
Yokosuka
Osamu
O
Omata
Masao
M
D020816:Amino Acid Motifs / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000821:virology*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D015342:DNA Probes; D004279:DNA, Viral; D019914:Taq Polymerase