Sanger测序法在乙型肝炎患者耐药突变检测中的应用

Application of Sanger sequencing in detection of drug-resistant mutations in patients with hepatitis B

目的分析Sanger测序在乙型肝炎病毒(HBV)耐药和基因型检测中的应用,并进一步探讨替比夫定(LdT)、阿德福韦(ADV)、拉米夫定(LAM)、恩曲他滨(FTC)、替诺福韦(TDF)以及恩替卡韦(ETV)6种核苷酸药物耐药发生的情况,为临床用药提供参考。方法收集中日友好医院215例HBV-DNA拷贝数大于103的乙型肝炎患者血清,提取血清中的HBV-DNA,采用聚合酶链式反应(PCR)方法进行扩增,并对PCR产物进行Sanger测序,使用STANFORD软件分析比对测序结果。并采用SPSS17.0软件进行统计学处理。结果本研究中,共有40例(18.6%,40/215)患者检测到发生耐药基因突变,其中具有对应参考价值的为21例(9.77%,21/215)。这215例患者中相关药物耐药突变发生率从高至低依次是LdT(9.77%,21/215)、FTC(7.44%,16/215),ETV(6.98%,15/215),LdT(6.05%,13/215),ADV(0.93%,2/215);未检出与TDF耐药相关突变。215例患者中C基因型发生率明显高于B基因型(P<0.01),C基因型与B基因型基因突变情况差异无统计学意义(P>0.05)。结论该研究提示Sanger测序可以较全面地分析乙型肝炎耐药基因突变情况;并且该研究也提示LAM药物作为慢性乙型肝炎(CHB)治疗首选药物的高耐药发生性。

Objective To analyze the application of Sanger sequencing in the detection of hepatitis B virus drug resistance and genotype,and to further explore the occurrence of six nucleotide drug resistance in hepatitis B,including tibivudine(LdT),adefovir(ADV),lamivudine(LAM),entracycline(FTC),tenofovir(TDF)and entecavir(ETV).To provide reference for clinical medication.Methods The serum samples of 215 patients with HBV-DNA copies greater than 10 3 in China-Japan Friendship Hospital were collected.The HBV-DNA in serum was extracted and amplified by PCR.The PCR products were sequenced by Sanger sequencing.The results were analyzed and compared by STANFORD software.SPSS17.0 software was used for statistical analysis.Results A total of 40 patients(18.6%,40/215)were found to have drug resistance gene mutations,of which 21(9.77%,21/215)had corresponding reference value.Tibivudine(9.77%,21/215),entecavir(7.44%,16/215),entecavir(6.98%,15/215),tibivudine(6.05%,13/215)and adefovir(0.93%,2/215)were the most common drug-resistant mutations in the 215 patients.The incidence of genotype C was significantly higher than that of genotype B in 215 patients(P<0.01).There was no significant difference between genotype C and genotype B(P>0.05).Conclusion This study suggests that the mutation of HBV resistance gene can be analyzed comprehensively by Sanger sequencing,and this study also suggests the occurrence of high drug resistance of LAM drugs as the first choice for the treatment of chronic hepatitis B(CHB).