Im Neuenheimer Feld, Heidelberg, Germany.
N Biotechnol. 2010 May 31;27(2):149-55. doi: 10.1016/j.nbt.2010.03.005. Epub 2010 Mar 30.
A strategy allowing for amplification, detection and genotyping of different genomic DNA targets in a single reaction container is described. The method makes use of primer-directed solution-phase amplification with integrated labeling in a closed, microfluidic oligonucleotide array. Selective array probes allow for subsequent detection and genotyping of generated amplicons by hybridization. The array contains up to 15,624 programmable features that can be designed, de novo synthesized and tested within 24 hours using an automated benchtop microarray synthesizer. This enables rapid prototyping and adaptation of the system to newly emerging targets such as pathogenic bacterial or viral subtypes. The system was evaluated by amplifying and detecting different loci of viral (HPV), bacterial (Bacillus sp.) and eukaryotic (human) genomes. Multiplex PCR and semi-quantitative detection with excellent detection limits of <100 target copies is hereby demonstrated. The high automation grade of the system reduces contamination risk and workload and should enhance safety and reproducibility.
本文描述了一种在单个反应容器中扩增、检测和基因分型不同基因组 DNA 靶标的策略。该方法利用引物指导的溶液相扩增，并在封闭的微流控寡核苷酸阵列中进行集成标记。选择性的阵列探针允许通过杂交对生成的扩增子进行后续检测和基因分型。该阵列包含多达 15624 个可编程特征，可以在 24 小时内使用自动化台式微阵列合成仪进行设计、从头合成和测试。这使得系统能够快速原型设计，并适应新出现的目标，如致病性细菌或病毒亚型。该系统通过扩增和检测病毒（HPV）、细菌（芽孢杆菌属）和真核（人类）基因组的不同基因座进行了评估。通过该系统进行的多重 PCR 和半定量检测显示出优异的检测限，<100 个目标拷贝。该系统的高自动化程度降低了污染风险和工作量，应提高安全性和重现性。
