Martin C S, Butler L, Bronstein I
Tropix, Bedford, MA, USA.
Biotechniques. 1995 May;18(5):908-13.
Quantitative PCR and reverse transcription PCR (RT-PCR) are widely used in biomedical, industrial and other research applications to determine the number of RNA or DNA molecules of a specific type and/or sequence in a sample of interest. We have developed an assay system to accurately quantitate PCR products that utilizes solid-phase capture and an enzyme-linked chemiluminescent detection method. The entire assay is performed in a single tube or microplate well. Biotinylated PCR products are quantitated by capture onto a streptavidin-coated surface, followed by hybridization of an internal fluorescein-labeled oligonucleotide probe and subsequent detection with an anti-fluorescein-alkaline phosphatase conjugate and CSPD chemiluminescent substrate. Light signal is measured in a luminometer. The assay sensitivity enables accurate quantitation of target DNA because the measurement is performed on product generated during the exponential phase of amplification. The broad dynamic range of the assay, which is greater than three orders of magnitude of PCR product concentration, simplifies the determination of the number of amplification cycles necessary for accurate quantitation of target molecules. The PCR-Light system is an ultrasensitive, non-isotopic and rapid assay for PCR product detection that also has general application to solution hybridization assays and other quantitation methods.
定量聚合酶链反应（PCR）和逆转录聚合酶链反应（RT-PCR）在生物医学、工业及其他研究应用中被广泛用于确定感兴趣样本中特定类型和/或序列的RNA或DNA分子数量。我们开发了一种检测系统，该系统利用固相捕获和酶联化学发光检测方法来准确定量PCR产物。整个检测在单个试管或微孔板孔中进行。生物素化的PCR产物通过捕获到链霉亲和素包被的表面进行定量，随后与内部荧光素标记的寡核苷酸探针杂交，并用抗荧光素-碱性磷酸酶偶联物和CSPD化学发光底物进行后续检测。在发光计中测量光信号。该检测的灵敏度能够准确地定量目标DNA，因为测量是在扩增指数期产生的产物上进行的。该检测具有宽泛的动态范围，大于PCR产物浓度的三个数量级，简化了准确定量目标分子所需的扩增循环数的确定。PCR-光系统是一种用于PCR产物检测的超灵敏、非同位素且快速的检测方法，也普遍适用于溶液杂交检测和其他定量方法。
