| | | 双内标PCR
| | 药学论文下载【关键词】 聚合酶链反应；,,酶联免疫吸附测定；,,基因定量；,,HIV
Establishment of a quantitative PCRELISA detection system with double internal standard
【Abstract】 AIM: To establish a quantitative PCRELISA detection system with double internal standard. METHODS: We designed one pair of primers (downstream primer was modified with DIG at its 5′ end) to amplify a fragment in HIV1 gag region and 2 samelength mutant fragments as internal standard, respectively. Then we designed 3 capture probes (they were modified with Biotin at its 5′ end) which could be hybridized with those 3 competitive PCR products respectively. We amplified the 3 templates of known amount and different concentrations at the same efficiency and in the same tube. Since they were marked, we could analyze them by ELISA and calculate the DNA copies of HIV1 per volume in each sample by formula. RESULTS: There was a fine linear relationship between the copies of HIV1 in fact and in the calculation through formula, with the correlation coefficient being 0.9998, the coefficient of variability being 9.48% in groups,and the error in average being 12.58%. No nonspecific amplification was observed. CONCLUSION: A quantitative PCRELISA detection system with double internal standard has been established successfully. The method is specific, sensitive and in low cost for clinical application.
【Keywords】 PCR； ELISA； gene quantification； HIV1
【摘要】 目的药学论文下载：建立双内标PCRELISA定量检测方法，并进行标定评价. 方法：制备了两种与HIV1野生扩增片段等长的药学论文下载PCR 内参照HS和LS，设计了一对HIV1 基因组gag区基因扩增引物,下游引物的5′端用地高辛修饰,可同时扩增115 bp左右的野生型和两种内参照片段. 设计三套捕获探针，其5′端用生物素（Biotin）修饰，可分别与竞争PCR 产物中的野生片段和两种突变型片段杂交. 同一反应管内,加入已知量但不同浓度的两种内参照及待测HIV1 DNA标准品, 它们将以相同的效率在同一个PCR管内被同时扩增并标记,酶联杂交法进行产物分析，结果用公式计算得出每单位体积待测标本中HIV1的DNA含量. 结果：公式计算出的HIV1拷贝数与实验加入的HIV1拷贝数这两组变量具有良好的线性关系，相关系数0.9998，批内变异系数9.48％，结果误差平均在12.58％,无非特异性扩增. 结论：建立了双内标PCRELISA定量检测方法，该方法具有特异、灵敏、成本低等特点，利于在临床实验上推广.
【关键词】 聚合酶链反应； 酶联免疫吸附测定； 基因定量； HIV1
0 引言
PCR技术已经十分普及，但是其自身具有的高灵敏度的特点使得实验中常常出现假阳性［1］. 酶联免疫吸附试验（enzymelinked immunosorbent assay, ELISA）法具有灵敏、特异、简单、快速、稳定等特点. 融合了二者优点的PCRELISA被广泛应用于临床检测领域，作为定量实验的效果很好［2］. 我们以HIV1前病毒的检测为模型，通过改进，建立了PCRELISA双内标（double internal standard）定量检测方法.
1 材料和方法
1.1材料
商品化的链亲和素（Avidin）预包被的96孔微孔检测板（Pierce公司，Cat. No. 15500）；10×PCR buffer，dNTP mix，MgCl2（Promega公司）；Taq酶由本室自制；AntiDIGPOD （Roche公司，Cat. No. 1207733）；显色底物TMB（Sigma公司）；GeneQuant pro微量紫外分光光度计（Amersham公司）；TGRADIENT PCR仪（Biometra公司）；Elx800型酶标仪（BioTek公司）；健康人血样6例、非HIV感染血样30例（甲肝、乙肝、单纯苞疹病毒、巨细胞病毒等）取自福州市第二医院；HIV感染血样4例取自福建省卫生防疫站.
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