乙型肝炎病毒YMDD变异与肝功能损伤程度的关系  

The study of the relationship between YMDD mutation and the degree of liver function destroyed

作　　者：王海滨[1] 姜平[1] 马洪滨[1] 王雪飞[1] 鞠连才[1] 徐军[1] 杨丽华[1] 刘立明[1] 李波[1] 李潇潇[1] 

机构地区：[1]解放军第302医院临床检验中心,北京100039

出　　处：《中华检验医学杂志》2005年第9期922-924,共3页Chinese Journal of Laboratory Medicine

摘　　要：目的采用自行研制的“微量核酸释放剂(micronucleicacidreleasingreagent,MNRR)”,建立微量血清直接进行PCR扩增的“一步法”实时荧光PCR检测拉米夫定治疗后YMDD变异的情况,探讨乙肝病毒YMDD变异与肝脏功能损伤程度的关系。方法设计包含YIDD和YVDD变异区和无变异参照的三条下游引物,与同一上游引物和探针建立3管荧光PCR扩增体系,变异Ct值与参照Ct值之差在3.3之内判断为阳性变异。建立将血清标本直接加到含有MNRR的扩增管进行核酸处理和PCR扩增的“一步法”检测。将0.2ml的PCR扩增管按3管/份标本排放到核酸提取仪,每管加入3.5μl的MNRR和3.5μl待测血清,用带虑芯吸头的加样器轻轻吹打混匀,加30μl无菌石蜡油覆盖,于80℃静置8min和10℃静置1min,直接加入PCR扩增液,封盖后移至实时荧光PCR仪进行扩增。对154例采用拉米夫定治疗1年后的乙肝患者YIDD变异发生率进行分析,并研究其与肝脏功能损伤程度之间的关系。结果拉米夫定治疗1年后YIDD变异率为15.9%;YVDD变异率为9.6%,YIDD和YVDD共生变异率为4.4%。发生YIDD/YVDD共生变异的患者其血清HBVDNA含量显著高于单一发生YIDD变异的患者(P<0.05),发生YMDD共生变异的乙肝患者其肝脏功能损伤的程度明显较单一发生YIDD变异者严重。结论拉米夫定治疗发生共生变异的患者其血清HBVDNA含量显著高于单一发生YIDD或YVDD变异的患者;发生YMDD共生变异的乙肝患者其肝功能损伤的程度明显重于无变异或单一发生YMDD变异的患者。Objective To develop an ONE STEP method of real-time PCR for detecting HBV DNA mutation of YMDD using Micro-Nucleic acid Releasing Reagent （MNRR） and to study the relationship between YMDD mutation and the degree of liver function destroyed. Methods The fluorescent probes and primers for mutation detection on the SLAN or PE 5700 were designed. The same HBV positive sense primer and TaqMan probe were used in three tubes each including antisense primers of control, YVDD and YIDD sequences. Less than 3. 3 Cts of YIDD or YVDD comparing with control test performed as the positive mutation. ONE STEP assay method of real time fluorescent PCR for YMDD has been developed as follows, 4 MNRR mixed mildly with 4 μl detection serum covered with 30μl mineral oil, incubated at 80℃ for 8 rain and 10℃ for 1 rain in nucleic acid releasing machine with the tube open. 45μl of Fluorescent-PCR reagents mixed in super clean bench was directly added to PCR amplification tube, and followed real-time PCR procedure： after one cycle at 95℃ for 2 rain, 53℃ for 30, followed by 39 cycles of second round PCR at 95℃ for 20 sec and 53℃ for 30 sec. 154 cases of hepatitis B treated with lamivudine for one year or more as Ct （cycle time ） , ALT, CHE and BILI of those cases were assayed by the auto CHE-machine of AU 5400. Results Data acquisition and analysis performed with PE 5700 express the mutation rate of YIDD was 15.9%, the YVDD was 9. 6% and the rate of cases in YIDD and YVDD simultaneously was 4. 4%. The concentrations of HBV DNA in patients with both YIDD and YVDD were significantly higher than that of patients only existing in mutation of YIDD. The activity of liver function of CHE and BILI destroyed degree in both YVDD and YIDD mutant cases was heavier than that of only YVDD or YIDD mutant cases. Conclusion The co-mutation with YIDD and YVDD in lamivudine treatment patients may be due to severe HBV replication rebounded. The activity of liver function of cholinesterase （CHE） and Bilirubin （BILI） destroyed

关 键 词：肝炎病毒 乙型 DNA突变分析 肝功能试验 乙型肝炎病毒YMDD变异 肝功能损伤 损伤程度 实时荧光PCR检测 HBVDNA含量 拉米夫定治疗 PCR扩增 

分 类 号：R446.5[医药卫生—诊断学]