用套式基因扩增技术检测乙型肝炎病毒P基因区YMDD变异

Detection of YMDD mutation in HBV polymerase gene region with nested PCR

目的 了解套式基因扩增 (nestedPCR)技术在检测HBVP基因区酪氨酸 -蛋氨酸 -天冬氨酸 -天冬氨酸 (YMDD)变异中的敏感性和特异性。方法 用套式基因扩增技术对 3组共 85例慢性乙型肝炎患者血清进行YMDD变异检测 ,检测结果经琼脂糖凝胶电泳判断。第 1组 :4 0例未经拉米夫定治疗 ,HBVDNA >5× 10 5拷贝 /ml;第 2组 :2 4例经拉米夫定治疗 6～ 18个月 ,HBVDNA≤ 5× 10 5拷贝 /ml;第 3组 :2 1例经拉米夫定治疗 6～ 18个月 ,HBVDNA >5× 10 5拷贝 /ml。结果 第 1组检出YVDD 2例、YVDD +YIDD 1例 ,检出率为 7.5 % ;第 2组检出YVDD、YIDD各 1例 ,检出率为 8.3% ;第 3组检出YVDD 4例、YIDD 2例、YVDD +YIDD 3例 ,检出率为 4 2 .9%。结论 套式基因扩增技术检测YMDD变异具有较高的敏感性和特异性 ,适合变异株快速检测 。

Objective To study the sensitivity and specificity of nested PCR for detection of YMDD mutation. Methods 85 serum samples from patients with chronic hepatitis B were divided in 3 groups: (1) 40 cases not treated with lamivudine, HBV DNA>5×10 5 copies/ml. (2) 24 cases treated with lamivudine for 6-18 months, HBV DNA ≤5×10 5 copies/ml. (3) 21 cases treated with lamivudine for 6-18 months, HBV DNA >5×10 5 copies/ml. Serum HBV DNA from these patients were amplified with nested PCR and then analyzed by electrophoresis. Results In the first group there were 2 YVDD mutation strains and 1 YVDD/YIDD mixed mutation strain, the rate of YMDD mutation was 7.5%. In the second group there was 1 YVDD and 1 YIDD mutation strains, the rate of YMDD mutation was 8.3% . In the third group there were 4 YVDD, 2 YIDD and 3 YVDD/YIDD mixed mutation strains, the rate of YMDD mutation was 42.9%. Conclusions The nested PCR is a high specific, sensitive and rapid method for detecting HBV YMDD mutation, especially for measurement of HBV YMDD mutation strains at the lower level.