变性高效液相色谱法检测乙型肝炎病毒YMDD基序的变异

Utilization of Denaturing High Performance Liquid Chromatography in Analysis of YMDD Motif Mutations of Hepatitis B Virus

目的建立变性高效液相色谱(DHPLC)检测HBV DNA聚合酶活性区酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸(YMDD)基序变异的方法。方法分别采用荧光PCR法和DHPLC法检测61例慢性乙型肝炎(CHB)患者经拉米夫定治疗1年后HBV YMDD基序变异,并经基因测序法验证。结果共检出10例野生型、5例DNA聚合酶活性区酪氨酸-异亮氨酸-天门冬氨酸-天门冬氨酸(YIDD)完全突变型、2例DNA聚合酶活性区酪氨酸-缬氨酸-天门冬氨酸-天门冬氨酸(YVDD)完全突变型,3例YMDD与YIDD混合突变型,1例YMDD与YVDD混合突变型,2种方法检出结果一致;21例PCR产物经过基因测序法得到证实。结论PCR结合DHPLC检测YMDD基序变异,经济便捷,适用于筛查CHB患者的YMDD基序变异。

Objective To use the method of denaturing high performance liquid chromatography （DHPLC） for the detection of YMDD motif mutations. Methods YMDD motif mutations in HBV were detected by fluorometric real-time PCR and DHPLC, and confirmed by analysing their gene sequence. Results Among 61 patients with chronic hepatitis B receiving one year therapy with lamivudine, 21 HBV DNA polymerase active motif sequence were found, contains 10 widetype YMDD, 5 cases of YIDD, 2 cases of YVDD, 3 cases of mixture mutation of YMDD an YIDD, 1 case of mixture mutation of YMDD and YVDD. All PCR products were identified by gene sequence determination. Conclusion The combination of routine PCR and DHPLC might be suitable for the screening of HBV YMDD motif mutation.