摘要
目的:建立双内标PCR-ELISA定量检测方法,并进行标定评价.方法:制备了两种与HIV-1野生扩增片段等长的PCR内参照HS和LS,设计了一对HIV-1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115bp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HIV-1DNA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HIV-1的DNA含量.结果:公式计算出的HIV-1拷贝数与实验加入的HIV-1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PCR-ELISA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.
AIM： To establish a quantitative PCR-ELISA 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 HIV-1 gag region and 2 same-length 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 HIV-1 per volume in each sample by formula. RESULTS： There was a fine linear relationship between the copies of HIV-1 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 PCR-ELISA detection system with double internal standard has been established successfully. The method is specific, sensitive and in low cost for clinical application.
出处
《第四军医大学学报》
CAS
北大核心
2007年第7期666-669,共4页
Journal of the Fourth Military Medical University
基金
福建省科技攻关重点项目(2001Z041)
