Objective: To analyse the emergence of YMDD motif(tyrosine-methionine-aspartate-aspartate) variants inpatients with hepatitis B treated with lamivudine.Methods: The amino acid substitution from methio-nine or isoleuci...Objective: To analyse the emergence of YMDD motif(tyrosine-methionine-aspartate-aspartate) variants inpatients with hepatitis B treated with lamivudine.Methods: The amino acid substitution from methio-nine or isoleucine at the YMDD motif at the HBVpolymerase gene is a main mutation resistant to lami-vudine treatment. Generated from a fragment of do-main C of the polymerase gene, patients HBV DNA,which had been positive previously became positive a-gain ever since it had been negative during lamivudi-ne therapy. Variants were detected by cleavage of theproducts of the three PCRs with following enzymes:FokI, SspI, Alw441. The results of PCR-RELP wereanalysed by 8. 4% polypropylene acidemide gel elec-trophoresis. PCR-RFLP assay was compared to di-rect sequencing.Results: HBV DNA was positive again in 33 patientsand positive for one year in 2 patients. YMDD vari-ants were detected in serum 14 of 35 patients, YIDDvariants in 4, YVDD variants in 6, and YI/MDD va-riants in 1; all were in concordance with the resultsof direct sequencing. The samples of other 3 patientsshowed YI/VDD mutations, as shown by direct se-quencing. The results of PCR-RFLP assay of themixed sera of YIDD and YVDD variants were similarto those sera of YI/VDD variants.Conclusion: PCR-RFLP is suitable for rapid detec-tion of YMDD variants of viral polymerase in hepati-tis B virus patients treated with lamivudine.展开更多
文摘Objective: To analyse the emergence of YMDD motif(tyrosine-methionine-aspartate-aspartate) variants inpatients with hepatitis B treated with lamivudine.Methods: The amino acid substitution from methio-nine or isoleucine at the YMDD motif at the HBVpolymerase gene is a main mutation resistant to lami-vudine treatment. Generated from a fragment of do-main C of the polymerase gene, patients HBV DNA,which had been positive previously became positive a-gain ever since it had been negative during lamivudi-ne therapy. Variants were detected by cleavage of theproducts of the three PCRs with following enzymes:FokI, SspI, Alw441. The results of PCR-RELP wereanalysed by 8. 4% polypropylene acidemide gel elec-trophoresis. PCR-RFLP assay was compared to di-rect sequencing.Results: HBV DNA was positive again in 33 patientsand positive for one year in 2 patients. YMDD vari-ants were detected in serum 14 of 35 patients, YIDDvariants in 4, YVDD variants in 6, and YI/MDD va-riants in 1; all were in concordance with the resultsof direct sequencing. The samples of other 3 patientsshowed YI/VDD mutations, as shown by direct se-quencing. The results of PCR-RFLP assay of themixed sera of YIDD and YVDD variants were similarto those sera of YI/VDD variants.Conclusion: PCR-RFLP is suitable for rapid detec-tion of YMDD variants of viral polymerase in hepati-tis B virus patients treated with lamivudine.
