实时荧光PCR检测乙肝病毒YMDD变异的临床应用

Use of Real-time PCR to Detect Mutations in the YMDD Region of the HBV Polymerase

目的 :探讨实时荧光 PCR检测 HBV YMDD变异价值。方法 :比较实时荧光 PCR法与 PCR产物直接测序的结果 ,以评价该方法的准确性 ,同时测定 10 3例拉米夫定治疗不同时期的患者血清 ,分析各时期的变异率及临床资料。结果 :19例标本的实时荧光 PCR结果与 PCR产物测序结果完全一致。10 3例患者中 13例发生 YMDD变异 ,治疗 7个月～ 12个月 YMDD变异率为 15 .2 % (5 /33) ,治疗 1a以上 YMDD变异率为 33.3% (8/2 4 )。变异患者与非变异患者体内的病毒量差异无显著性 ;变异患者 AL T异常发生率高于非变异患者 ,但升高程度不如部分非变异患者 ;变异患者保护性抗体的出现率较低。结论 :实时荧光 PCR检测 YMDD变异是一种准确、灵敏、经济、简便。

Objective To evaluate the value of real-time PCR and melting curve, in detecting mutations in the YMDD region of the HBV polymer ase.Methods Evaluate the accuracy of real-time PCR by comparing it s results with that by direct sequencing to the products of PCR. Also analyze ser um of 103 patients with chronic hepatitis B and analyze the occurrence of differ ent phases of therapy.Results The results of 19 samples by two met hods are all the same. And thirteen patients in 103 are found to have mutation s in the YMDD region of HBV polymerase. The occurrence of variants in patients aft er treatment 7 to 12 months with lamivudine is 15.2 %( 5/33), and that in patien ts after treatment more than a year with lamivudine is 33.3 %( 8/24). There is n o significance of the quantitative of HBV between patients with and without muta tions in the YMDD region of HBV polymerase. The occurrence of abnormal ALT in pa tients with mutations in the YMDD region of HBV polymerase is higher than that i n patients without mutations in the YMDD region of HBV polymerase, and ALT level in patients with YMDD variants are not as high as that in part of patients with out mutations in the YMDD region of HBV polymerase. The occurrence of protective antibodies against HBV is low in patients with mutations in the YMDD region of HBV polymerase.Conclusion Real-time PCR provides a rapid, accurat e , economic, sensitive way to detect lamivudine resistance-associated mutations i n the YMDD region of the HBV polymerase, and it is suitable for handling batches of samples.