实时荧光PCR法快速检测乙型肝炎病毒基因YMDD突变株

Rapid Detection of Hepatitis B Virus YMDD Gene Variant by Real-time Fluorescent PCR

目的建立一种敏感、特异、快速的实时荧光PCR定量检测乙型肝炎病毒基因YMDD突变株的方法,为临床治疗提供指导。方法设计引物和TaqMan-MGB探针,利用TaqMan-MGB探针技术,建立实时荧光定量PCR法。检测临床HBV-YMDD变异标本36份和野生型HBV标本20份,并将其YMDD变异结果与DNA测序结果进行比较。结果该方法检测灵敏度为101拷贝/μl;特异性为100%;最低检测限度为101DNA拷贝/30μl反应体系;实时荧光PCR方法测得YMDD野生株20份,变异株36份,与DNA测序结果完全一致,符合率为100%。结论应用TaqMan-MGB探针的实时荧光定量PCR方法检测YMDD变异株基因,具有灵敏、特异和精确等优点,对临床监测拉米夫定耐药具有重要意义。

Objective To develop a sensitive, specific and rapid real-time fluorescent PCR for detection of hepatitis B （HB） virus YMDD gene variant and provide a basis for clinical treatment of HB. Methods Develop a real-time fluorescent PCR by using designed primers and TaqMan-MGB probe. Thirty-six chnical specimens of HBV-YMDD variants and 20 specimens of wild type HBV were detected by the developed real-time fluorescent PCR, and the results were compared with those of DNA sequencing. Results The sensitivity, specificity and minimum detection limit of the developed real-time fluorescent PCR were 10^1 copies/μl, 100% and 10^1 DNA copies/30μl reaction system respectively The detection result of developed PCR was completely consistent with that of DNA sequencing. Conclusion The developed real-time fluorescent PCR was sensitive, specific and accurate for the detection of HB virus YMDD gene variant and of important significance in clinical monitoring of resistance to lamivudine.