Verhaegen M, Christopoulos T K
Department of Chemistry, University of Windsor, Ontario, Canada.
Anal Chem. 1998 Oct 1;70(19):4120-5. doi: 10.1021/ac980512k.
We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24-bp sequence, centrally located. Biotinylated PCR products from target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe, and the hybrids were determined by using antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a fluorescein-labeled probe, and the hybrids were determined by using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was measured by adding a Ca(2+)-containing light-triggering solution. Alkaline phosphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430 to 315,000 target DNA molecules. Average CVs ranged from 7 to 17%. The proposed system is expected to facilitate the automation and routine use of quantitative PCR.
我们开发了一种用于定量聚合酶链反应（PCR）的双分析物化学发光杂交检测方法。该方法能够在同一反应容器中同时测定扩增的靶DNA和内标（IS）。来自样品的靶DNA（233 bp）与恒定数量的重组DNA IS共同扩增，该IS与靶DNA具有相同的大小和引物结合区域，仅在位于中心位置的24 bp序列上有所不同。靶DNA和IS的生物素化PCR产物捕获在涂有链霉亲和素的单个微量滴定孔上。扩增的靶DNA与地高辛配体标记的特异性探针杂交，通过使用水母发光蛋白标记的抗地高辛配体抗体测定杂交体。扩增的DNA IS在同一孔中与荧光素标记的探针杂交，通过使用与碱性磷酸酶偶联的抗荧光素抗体测定杂交体。通过添加含钙的光触发溶液来测量水母发光蛋白。通过使用二氧杂环丁烷化学发光底物来测量碱性磷酸酶。从靶DNA和IS扩增产物获得的发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。线性范围从430至315,000个靶DNA分子。平均变异系数范围为7%至17%。预计所提出的系统将有助于定量PCR的自动化和常规使用。
