Philips Research, High Tech Campus 11, 5656 AE Eindhoven, The Netherlands.
Lab Chip. 2012 Apr 24;12(10):1897-902. doi: 10.1039/c2lc20740k. Epub 2012 Apr 3.
Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.
实时荧光定量聚合酶链反应（qrtPCR）被广泛用作研究和诊断工具。尽管该方法具有许多强大的功能，但由于可用荧光团的光谱重叠，其多重检测的程度受到限制，大约为 6。本文提出了一种新方法，通过整合溶液中的扩增和实时杂交到微阵列进行监测，允许以更高的多重化进行定量扩增检测。该方法不需要对 PCR 产物进行任何操作，并且在单个封闭室中运行。采用标记引物，主要挑战之一是针对溶液中的高荧光背景来测量表面信号。使用基于 DVD 技术的微型光学器件和与平板热循环仪相结合的紧凑型共焦扫描仪，实现了同时扫描和加热。在单重反应中，该方法的可行性得到了证明，其分析灵敏度可与常规 qrtPCR 相媲美。
