乙型肝炎病毒基因型、拉米夫定耐药突变的RFLP检测及临床相关性分析

Determination of hepatitis B virus genotypes and lamivudine resistant mutations by RFLP

目的对乙型肝炎(简称乙肝)肝病毒进行基因分型,检测患者服用拉米夫定后血清中乙肝病毒突变情况,并进行临床相关性分析。方法设计乙肝病毒突变及基因分型特异性引物和酶切位点,系用限制性片断长度多态性(RFLP)分析法检测突变类型并对乙肝病毒进行基因分型。结果在115份被检测的血清标本中,75份(65%)标本为野生型,18份(15.7%)标本为YIDD突变,10份(8.7%)标本为YVDD突变;在混合型突变中,YIDD+YVDD存在于6份(5.2%)标本中,YMDD+YIDD存在于4份(3.5%)标本中,2份(1.7%)标本为YMDD+YVDD混合突变型;未检测到YSDD突变型。在180位点的突变检测中,98份(85%)标本为野生型,5份(4.3%)标本为rtL180M,L180M+M204I存在于8份(6.96%)标本中,另4份(3.5%)标本为L180M+M204V混合突变型。在115份标本中,102份标本为C基因型,13份标本为B基因型,未发现其他基因型。通过χ2检验,乙肝病毒YMDD突变与患者的性别、血清DNA水平、病毒基因型之间没有相关性(P>0.05),而乙肝病毒YMDD变异与患者服用拉米夫定的时间长短有相关性(P<0.05),用药时间越长,发生突变的几率越大。乙肝患者服用拉米夫定后,180位点的突变和204位点突变具有相关性(P<0.05)。结论采用RFLP分析法可以有效地检测乙肝病毒YMDD突变并对乙肝病毒进行基因分型。

Objective To detect hepatitis B virus （HBV） genotypes and the effect of lamivudine on HBV muta- tions. Methods Restriction sites and special primers for determination of HBV mutations and HBV genotypes were designed. HBV mutations and genotypes were determined by using RFLP. Results In the 115 samples, 75（65%） were YMDD, 18（15.7%） were YIDD, 10（8.7%） were YVDD, 6 （5.2%）were YIDD ＋ YVDD, 4（3.5%） were YMDD＋ YIDD and 2 （1.7%） were YMDD ＋ YVDD; none were YSDD. At codon 180, 98 samples were wild types, 5（4.3% ） were rtL180M,8（6.96%） were L180M ＋ M204I, and 4（4.3%）were L180M ＋ M204V. In 115 samples, 102 samples were genotype C and 13 samples were genotype B. The resuhs of X2 test showed that the rate of lamivudine resistant mutations might not have a positive correlation with gender, HBV DNA levels and genotype （ P 〉 O. 05）, however, they might have a positive correlation with time of lamivudine therapy （ P 〈 0.05）, and the rate of lamivudine resistant mutations increased with prolonged lamivudine therapy. Also, mutation at cedon 180 might have a positive correlation with that at cedon 204 after lamivudine therapy（ P 〈 0.05）. Conclusion YMDD mutations of HBV and genotype can be effectively determined by RFLP.