不同实验方法在监测拉米夫定耐药突变中的应用价值  

Evaluation of different methods in monitoring YMDD motif mutations associated with lamivudine resistance

作　　者：胡盈莹[1] 江家骥[1] 李丹[1] 林彩文[1] 李勤光[1] 陈怡[1] 

机构地区：[1]福州市传染病医院,350025

出　　处：《中华肝脏病杂志》2003年第7期427-430,共4页Chinese Journal of Hepatology

摘　　要：目的 通过对三种检测乙型肝炎病毒(HBV)YMDD突变方法的比较,评价其在监测拉米夫定抗HBV治疗中发生耐药突变的价值。方法 用熔解曲线法、聚合酶链反应微板核酸杂交-酶联免疫吸附法(PCRmnh-ELISA)和错配聚合酶链反应限制性片段长度多态性分析法(mPCR-RFLP)对拉米夫定治疗过程中出现HBV DNA由阴转阳的44例慢性乙型肝炎患者的血清进行YMDD突变检测,比较它们的敏感性,并与直接测序法比较它们的特异性。结果 mPCR-RFLP法、PCRmnh-ELISA法和熔解曲线法检出HBV DNA的灵敏度分别为10~4、10~5和10~6拷贝/ml。44例患者血清中,用mPCR-RFLP法检出26例为YMDD突变株,18例为野生株。在26例突变株中,熔解曲线法检出16例,PCRmnh-ELISA法检出18例;而在18例野生株中,熔解曲线法检出2例突变株,PCRmnh-ELISA法检出13例突变株。将上述三种方法检测结果不一致的16例标本进行测序,mPCR-RFLP法、熔解曲线法和PCRmnh-ELISA与测序的符合率分别为93.8%(15/16)、43.8%(7/16)、18.8%(3/16),差异有极显著性(x^2=18.7,P<0.01)。结论 mPCR-RFLP法检测YMDD突变株具有较好的可靠性,是监测拉米夫定耐药株的一种非常有效的方法;熔解曲线法和PCRmnh-ELISA法需要进一步完善以提高敏感性和特异性。Objective To evaluate the three different methods in monitoring the lamivudine-resistant HBV mutants in lamivudine-treated patients with chronic hepatitis B. Methods The sensitivity and specialty of melting curve assay and polymerase chain reaction microplate nucleotide hybridization-enzyme linked immunosorbent assay (PCRmnh-ELISA) were compared with those of mismatch polymerase chain reaction-restriction fragment length polymorphism (mPCR-RFLP) and sequence analysis, through detection of HBV YMDD mutants in 44 serums from chronic hepatitis B patients receiving lamivudine monotherapy at the time of viral breakthrough. Results mPCR-RFLP assay was more sensitive (104 copies/ml) than both PCRmnh-ELISA (105 copies/ml) and melting curve assay (106 copies/ml). 26 YMDD mutants and 18 wild-types were determined by the means of mPCR-RFLP. Among the 26 mutants, only 16 and 18 mutants were found by melting curve assay and PCRmnh-ELISA, respectively. Whereas, out of the 18 wild-types, 2 and 13 mutants were detected by melting curve assay and PCRmnh-ELISA, respectively. To confirm the different results determined by the three methods in 16 samples, sequence analysis was conducted and showed that the rate of consistency with sequencing was 93.8% by mPCR-RFLP, 43.8% by melting curve, and 18.8% by PCRmnh-ELISA, respectively ( x2 = 18.7, P<0.01). Conclusions The mPCR-RFLP assay is reliable to monitor HBV YMDD mutantions. Melting curve assay and PCRmnh-ELISA should be further improved to increase their sensitivity and specialty.

关 键 词：实验方法 拉米夫定 耐药性 基因突变 YMDD HBV 

分 类 号：R512.62[医药卫生—内科学]