Published online Feb 28, 2006. doi: 10.3748/wjg.v12.i8.1308
Revised: October 15, 2005
Accepted: October 26, 2005
Published online: February 28, 2006
AIM: 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 ΔCt < 3.5 (ΔCt = 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 1 000 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.
CONCLUSION: 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.