Taqman MGB探针分型技术检测HBV YMDD 变异及临床应用研究

摘　要: 目的：建立一种快速准确的HBV YMDD变异检测方法，用于乙肝患者拉米夫定治疗中耐药性突变的监测。方法：采用三种特异性TaqmanMGB探针和一对共同引物，建立了检测HBV YMDD变异的荧光定量PCR方法。分别针对总HBV 、YIDD变异、YVDD变异的三管并行检测，可以确定YMDD变异的类型及其在病毒群中的比例。对YMDD野生株、YIDD变异株、YVDD变异株的病毒质粒经过梯度稀释进行检测验证。以序列直接测定法作为诊断YMDD 变异的金标准，对该方法YMDD变异检测的灵敏度、特异性和诊断符合率作评价。并对112例经拉米夫定治疗的血清HBV DNA持续阳性的乙型肝炎患者检测其YMDD变异情况。结果　该方法在105--108Copies/ml 的YMDD野生株、YIDD变异株、YVDD变异株检测中匀未出现非特异性扩增信号；相同浓度的YMDD、YIDD、YVDD在三种不同探针的反应管中的扩增效率基本一致；病毒野生株与YVDD变异株的实际混合比例与计算比例基本一致；该方法检测YIDD、YVDD的最低检测界限为1000 Copies/ml，能在106Copies/ml病毒群中检出0.1%的变异株的存在。以直接测序方法为金标准，该方法YMDD变异检测的灵敏度100%、特异性96%，诊断符合率97.5%。该方法检测112例经拉米夫定1--3年治疗血清HBV-DNA持续阳性的乙型肝炎患者37 例发生YMDD 变异,阳性率27.2%。结论：该方法能够直接检测HBV YMDD变异的类型及其在病毒群中的比例，具有快速、灵敏、准确、经济实用等优点，适合临床实验室开展拉米夫定耐药性突变的监测。

Taqman MGB probe type technology to detect HBV YMDD mutation and the clinical study.

Abstract: Objective: To establish a fast and accurate HBV YMDD mutation detection method for the treatment of hepatitis B patients with lamivudine resistance mutations in the monitoring. Methods: Three specific TaqmanMGB probe and a primer to establish a common HBV YMDD mutation detection of fluorescence quantitative PCR. Respectively for the HBV, YIDD variation, YVDD variation of the three parallel testing, to determine the type of YMDD mutation in the virus and its proportion in the group. YMDD wild strains, YIDD mutant, YVDD mutation of the virus after gradient plasmid diluted, for testing verification. As to direct sequencing of the gold standard to evaluate this method of YMDD mutations detection sensitivity, specificity and diagnostic rate. Detection of the 112 cases of lamivudine therapy serum HBV DNA continued positive hepatitis B patients YMDD variation. Results: The method in the 105 - 108 Copies / ml of YMDD wild strains, YIDD mutant, YVDD mutation of detection does not appear to absorb non-specific amplification signal; The same concentration of YMDD, YIDD, YVDD in three different reactions of the probe, The amplification efficiency was the same; Wild strains of the virus mixing with the mutant strain, the ratio of its actual proportion consistent with the calculation.The method of detection limits for the 1000 Copies / ml, the virus can be detected among the 0.1% of the mutation of existence. To direct sequencing approach to the standards, the YMDD mutations methods of detection sensitivity of 100%, specificity 96%, the diagnosis rate of 97.5 percent. The 112 cases were detected by lamivudine 1 - 3 years of sustained serum HBV-DNA-positive patients with hepatitis B YMDD mutation occurred in 37 cases, the positive rate of 27.2 percent. Conclusion: The method to directly detect HBV YMDD mutation of the virus and its type in the proportion of group, with rapid, sensitive and accurate, economical and practical advantages for clinical laboratories lamivudine-resistant mutations of the monitoring.