Establishment of a quantitative PCRª²ELISA detection system with double internal standard

    QIU Yuª²Miao, HUANG Yiª²De£¬ ZHANG Yanª²Ding

    1Department of Developmental Biology, College of Life Science, Fujian Normal University

    2Key Laboratory of Developmental Biology and Neurobiology, Fujian Institution for Higher Learning, Fuzhou 350007, China

    ¡¾Abstract¡¿ AIM: To establish a quantitative PCRª²ELISA detection system with double internal standard. METHODS: We designed one pair of primers (downstream primer was modified with DIG at its 5¡ä end) to amplify a fragment in HIVª²1 gag region and 2 sameª²length mutant fragments as internal standard, respectively. Then we designed 3 capture probes (they were modified with Biotin at its 5¡ä 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 HIVª²1 per volume in each sample by formula. RESULTS: There was a fine linear relationship between the copies of HIVª²1 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 PCRª²ELISA detection system with double internal standard has been established successfully. The method is specific, sensitive and in low cost for clinical application. ......