| Objective Lamivudine is a nucleoside analogue that is an effective inhibitor of the viral polymerase for hepatitis B virus (HBV).In clinical trials,lamivudine has been effective in reducing HBV DNA levels in the sera of patients infected hepatitis B virus.However,prolonged treatment with lamivudine for HBV infection can result in the emergence of YMDD motif(tyrosine-methionine-aspartate-aspartate)in region of the reverse transcriptase of HBV ,with the methionine(M) at HBV codon 552 replaced by isoleusine (I) or valine (V) .The emergence of YMDD mutif mutation can result in acute exacerbation in part patients during lamivudine therapy. Presently ,the detection of emerging HBV variants by direct sequencing of the HBV genome is excessively time-consuming and expensive for analysis of clinical samples.The aim of study was to analyse the emergence of lamivudine-resistant HBV genotypes by means of restriction fragment length polymorphism (RFLP) of the polymerase chain reaction(PCR) product generated from a fragment of domain C of the polymerase gene,which can detect rapidly and conveniently the YMDD variants in clinic.Theresults with the method were compared with those obtained with a direct sequence technique.Methods We collected the clinical samples as follows: patients's HBV DNA became positive again and they were negative even since duing lamivudine therapy, and patients's HBV DNA were persistly positive for one year during lamivudine therapy.The diagnosis of 35 patients as follows: 23 patients with chronic hepatitis B, 2 patients accepted liver transplantations ,9 patients with asymptomatic carriers and one patient with decompensative hepatic cirrhosis (B) accompanied with hypersplenism before treatment. Variants were detected by cleavage of the products of the three PCRs with following restriction enzymes:FokK SspK Alw441.The 274bp PCR fragments were digested with FokI .The 138bp PCR fragments were digested with Sspl.The 181bp PCR fragments were digested with Alw441.The results of PCR-RFLP were analysed by 8.4% polypropylene acidemide gel electrophoresis . The PCR-RFLP assay were compared with results direct sequencing.Result : Identification of YMDD variants. The 274bp PCR fragments of the wile-type YMDD and the mutant YVDD variant became 143bp and lOObp and 3 Ibp, those of the mutant YIDD variant became 243bp and 3Ibp by being digested with FokI. The 138bp PCR fragments of YIDD variant became 109bp and 29bp by digested with Sspl.but the Sspl can't cut the sequence of the mutant YVDD variant and wild-type YMDD.The 18Ibp PCR fragments of the mutant YVDD variant became 158bp and 23 bp by being digested with Alw441,but the Alw441 can't cut the sequence of the mutant YIDD variant and wild-type YMDD.The sequence of the mutant YI/VDD variants was partly cut by being digested with three restriction enzymes.The result of PCR-RFLP analysis were compared with those obtain with the direct sequencing technique.33 patients ' HBV DNA were positive the second time and wereever negative before.The HBV DNA in 2 patients were always positive for one year. 14 of 35 serum samples(40%) showed mutant in the YMDD sequence by PCR-RFLP assay, which were accorded with the results of direct sequening. Diagnosis of the 14 patients as follows:2 patients acceped liver transplantations ,9 patients with chronic hepatitis B and 3 patients with asymptomatic carriers before treatmemt.The sorts of YMDD variants were compared between the PCR-RFLP assay and the direct sequencing technique . YIDD variant in 4 cases and the YVDD variant in 6 cases and the YI/MDD in 1 case were dedected in 14 cases by the PCR-RFLP assay.HBV genotypes of 11 cases were confirmed by detected sequencing.The other 3 patients 'samples showed mixed mutations,namely both YIDD and YVDD variants.which compare to the direct sequence and were accorded with the results of direct sequencing too. The PCR-RFLP assay of the mixed sera of YIDD and YVDD variants are the same as that of the serum of YI/VDD variant.Conclued: (1) The PCR-RFL...
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