摘要
目的 建立血浆 (血清 )HBVDNA荧光定量PCR检测方法 ,探讨其临床应用价值。方法 用PUCm T载体和PCR纯化产物连接 ,转染DH5α菌 ,筛选阳性菌落 ,提取质粒 ,制作外标准品 ;在HBV基因组S区设计一对引物和TaqMan探针 ,严格优化反应物的组成和扩增条件 ,并对该方法进行评价。结果 建立的HBVDNA荧光定量PCR最低可检出 80 0拷贝 /ml;批内误差为 1 8%～ 6 5% ,批间误差为 2 6 %～ 9 1 % ;在 1 0 4 ～ 1 0 11拷贝 /ml之间与Ct值具有很好的线性 (Ct =- 1 .391 1ln(X) +4 1 .6 5,r =- 0 .9958) ;外周血HBVDNA临床定量检测发现 ,HBeAg阳性的 1 30例中 ,HBVDNA阳性率为 1 0 0 % ,含量为1 0 6 89± 1.6 9拷贝 /ml,抗HBe阳性的 96例中HBVDNA阳性率为 55 2 % ( 53/ 96 ) ,含量为 1 0 4 .0 9± 1.19拷贝 /ml;6 2例献血员未检出HBVDNA。乙肝临床治疗监测发现 ,外周血中HBVDNA含量检测可有效反映治疗效果 ,指导临床用药。结论 荧光定量PCR检测方法是一种相对准确、灵敏度高、特异性较强和较简便的HBVDNA定量检测技术 ,对乙肝诊断。
Objective To develop a fluorescence quantitative PCR based on TaqMan chemistry for quantification of HBV DNA in plasma .Methods A pair of primers were designed to amplify the fragment of 144 bp long from HBV S gene and a TaqMan probe of 20nt long between the primers,modified with 6 Fam at 5′ end and Tamra at its 3′ end,was designed to detect the PCR product during the PCR cycles.The experimetal conditions and reagents of amplification were sophisticatedly optimized in order to produce perfect amplificative efficiency and reduce non specific amplification.The PCR fragment of HBV DNA was cloned into vector PUCm T.The recombinant plasmid was purified and subsequently quantified as HBV DNA standard.Results The detectable limit of assay was 800 copies/ml.A linear standard curve was obtained between 10 4～10 11 copies/ml(Ct=-1.3911ln(x)+41.65, r = -0.9958 ).The coefficient of variation was 1.8%～6.5% and 2.6%～9.1% for intra and inter assay,respectively.HBV DNA was detected in 100%(162/162) of hepatitis B patients with positive HBeAg, and in 55.2%(53/96) with positive HBeAb,but was not detected in 62 cases of healthy donors.Meanwhile,quantification for HBV DNA in plasma was considered as a useful parameter for treatment of hepatitis B patients.Conclusion The FQ PCR for quantification of HBV DNA is a simple and less time consuming,highly sensitive and specific,reliable method.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2002年第5期282-284,共3页
Chinese Journal of Clinical Laboratory Science
基金
温州市"5 5 1"人才培养计划
温州市科委资助课题 (S2 0 0 1A49)
