Bortolin S, Christopoulos T K, Verhaegen M
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Anal Chem. 1996 Mar 1;68(5):834-40. doi: 10.1021/ac950898o.
Using a 308 bp DNA fragment (target DNA) as a template, we have synthesized an internal standard (IS) that is of the same size and uses the same primers as the target but differs by a 26 bp centrally located sequence. We then designed quantitative polymerase chain reaction (PCR) assays in which the target DNA is coamplified with a constant amount of IS (20,000 molecules). The presence of IS compensates for the reaction-to reaction variability of the amplification efficiency. The PCR products are assayed by two distinct hybridization protocols. The first approach (QPCR-1) requires that specific probes be immobilized onto microtiter wells, followed by hybridization with digoxigenin-labeled PCR product. In the second protocol (QPCR-2), PCR product is captured onto the wells and hybridized with digoxigenin-tailed specific probes. In both assays, the hybrids are detected using an antidigoxigenin-alkaline phosphatase conjugate and 5'-fluorosalicylphosphate as substrate. The hydrolysis product forms a highly fluorescent complex with Tb(3+)-EDTA, as measured by time-resolved fluorometry. The ratio of the fluorescence values obtained for the amplified target DNA and IS is linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear ranges are 1000-200,000 molecules for QPCR-1 and 2000-200,000 molecules for QPCR-2. The CVs ranged from 3.4 to 9.7%.
以一段308 bp的DNA片段（靶DNA）为模板，我们合成了一种内标（IS），其大小与靶标相同，使用相同的引物，但中心位置有一段26 bp的序列不同。然后我们设计了定量聚合酶链反应（PCR）检测方法，其中靶DNA与恒定数量（20,000个分子）的IS共同扩增。IS的存在补偿了扩增效率在反应之间的变异性。PCR产物通过两种不同的杂交方案进行检测。第一种方法（QPCR-1）要求将特异性探针固定在微孔板上，然后与地高辛标记的PCR产物杂交。在第二种方案（QPCR-2）中，PCR产物被捕获到孔上，并与地高辛尾特异性探针杂交。在这两种检测中，使用抗地高辛-碱性磷酸酶偶联物和5'-氟水杨酸磷酸酯作为底物来检测杂交体。水解产物与Tb(3+)-EDTA形成高度荧光复合物，通过时间分辨荧光法进行测量。扩增的靶DNA和IS获得的荧光值之比与扩增前样品中存在的靶DNA分子数量呈线性相关。QPCR-1的线性范围是1000 - 200,000个分子，QPCR-2的线性范围是2000 - 200,000个分子。变异系数范围为3.4%至9.7%。
