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  • Title: [Detection of YMDD mutations in hepatitis B virus DNA with the use of allele specific polymerase chain reaction].
    Author: Pichkovskaia VA, Fevraleva IS, Ibragimova MM, Solov'eva TI, Drel' PE, Sudarikov AB.
    Journal: Mol Gen Mikrobiol Virusol; 2004; (1):27-9. PubMed ID: 15025001.
    Abstract:
    Antiviral drug lamivudine has been widely used in the treatment of hepatitis B. However, it was demonstrated recently, that therapy by the drug is related with the selection of viral strains carrying mutations in C-domain of DNA polymerase/reverse transcriptase (YMDD-mutation). The mutation is the cause of resistance to lamivudine. An increase of the mutant population in the course of a long-term lamivudine therapy makes the monotherapy by the discussed drug ineffective. Therefore, the monitoring of YMDD mutations is important in the treatment of chronic hepatitis B (CHB) by lamivudine. The results of using the allele-specific polymerase chain reaction (AS PCR) for the detection of YMDD mutations are presented in the offered case study. Sera from CHB patients were used in research. A system of allele-specific primers was designed for the HBV DNA region from base 720 to base 978, which enabled us to detect possible substitutions in an appropriate ATG-triplet, i.e. the YMDD-mutations locus--positions 741-743 of the HBV DNA nucleotide sequence. The obtained DNA fragments were analyzed by electrophoresis in the agar gel. AS PCR was used to test the sera of HBV patients. Samples were detected, which contained the "wild" virus type, different YMDD mutations and mixed (the "wild" type plus a mutation) HBV variants. It was shown that reliable AS PCR results could be obtained only in those sample, whose HBV titer is no more than 10(6) genome per ml. The offered AS PCR procedure can be applied in the detection of YMDD mutations of HBV DNA in sera of patients with hepatitis B. Sera with a high virus titer must be diluted, prior to testing, to 10(6) genomes per ml. to ensure the reliable AS PCR results.
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