Detection of YMDD mutants using universal template real-time PCR.
OBJECTIVE
To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection.


METHODS
We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions (reaction V and reaction I) and total hepatitis B viruses were detected in another reaction for control (reaction C). Results were determined by deltaCt < 3.5 (deltaCt = Ct of reaction V or I - Ct of reaction C). Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing.


RESULTS
As many as 1000 copies per milliliter of wide-type plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples.


CONCLUSIONS
This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.
Jiangsu Oil Field Hospital, Shaobo, Jiangdu, China.
Rong-Sheng
RS
Wang
Hua
H
Zhang
Yu-Fen
YF
Zhu
Bei
B
Han
Zhi-Jun
ZJ
Yang
D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D001224:Aspartic Acid / Q000032:analysis; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology* / Q000627:therapeutic use; D008715:Methionine / Q000032:analysis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014443:Tyrosine / Q000032:analysis
D000998:Antiviral Agents; D004279:DNA, Viral; D019259:Lamivudine; D001224:Aspartic Acid; D014443:Tyrosine; D008715:Methionine