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Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR.

Abstract：

BACKGROUND/AIMS:Lamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge. Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene. We describe a method to detect and quantify mutant viral populations using amplification refractory mutation system (ARMS) PCR. METHODS:We developed a real-time ARMS-PCR to detect point mutations in the polymerase gene. Using real-time PCR (LightCycler) with a ResonSense probe, PCRs were performed using clones of the HBV polymerase gene containing the different YMDD mutations. Dilution series of the templates were made and tested against each of the primer pairs. This method was applied to quantify mutant virus in patient serum samples. RESULTS:As little as 0.01% mutant DNA in 10(5)-10(9) copies wild-type DNA were detected. The method is more sensitive than amplicon sequencing, which is the current method of mutant determination in the YMDD motif. CONCLUSIONS:This study demonstrates a rapid, highly sensitive and reproducible method of quantifying mutant HBV virus in lamivudine treated patients. It can be used to monitor patients before and during lamivudine therapy.

journal_name

J Hepatol

journal_title

Journal of hepatology

authors

Punia P,Cane P,Teo CG,Saunders N

doi

10.1016/j.jhep.2004.02.009

subject

Has Abstract

pub_date

2004-06-01 00:00:00

pages

986-92

issue

6

eissn

0168-8278

issn

1600-0641

pii

S0168827804000625

journal_volume

40

pub_type

杂志文章

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