BIOSYST-MeBioS, KU Leuven-University of Leuven, Willem De Croylaan 42, P.O. Box 2428, B-3001 Leuven, Belgium.
Anal Chem. 2013 Feb 5;85(3):1734-42. doi: 10.1021/ac303008f. Epub 2013 Jan 11.
A home-built fiber optic surface plasmon resonance platform (FO-SPR) was applied to directly screen PCR amplified DNA for mutations. The FO-SPR sensor was used for real-time monitoring of DNA duplex melting during high resolution temperature cycling. The signal of the DNA melting was enhanced by means of gold nanoparticle labels. This FO-SPR genetic assay allowed for detection of single-point mutations (SNP) in less than 20 min. The concept was demonstrated for the analysis of 9 different serogroups of the bacterium Legionella pneumophila, a common human pathogen responsible for atypical pneumonia. FO-SPR allowed us to detect genetic mutations inhibiting PCR, which could lead to amplification bias when molecular diagnostics are applied for L. pneumophila detection. All serogroups were found to display unique melting temperatures, indicating that mutations have accumulated in the target sequence. In a next step, clinical samples of L. pneumophila were analyzed using the FO-SPR sensor. This technology was proven to be reliable for the detection of mutations for those samples that previously displayed ambiguous qPCR quantification results. When these results were benchmarked, FO-SPR results were found to be consistent with Sanger sequencing but not with fluorescence based DNA melting. The presented results convincingly advocate the advantages of FO-SPR as a high resolution and fast genetic screening tool that can compete with the current standard techniques for SNP detection.
自行搭建的光纤表面等离子体共振平台（FO-SPR）被应用于直接筛选 PCR 扩增的 DNA 突变。FO-SPR 传感器用于在高分辨率温度循环过程中实时监测 DNA 双链体的解链。通过金纳米颗粒标记增强 DNA 解链的信号。这种 FO-SPR 遗传检测方法可以在不到 20 分钟的时间内检测到单点突变（SNP）。该概念已应用于分析细菌军团菌的 9 种不同血清型，军团菌是一种常见的人类病原体，可引起非典型肺炎。FO-SPR 允许我们检测抑制 PCR 的遗传突变，当应用分子诊断方法检测军团菌时，这可能导致扩增偏倚。所有血清型都显示出独特的解链温度，表明目标序列中已积累了突变。在下一步中，使用 FO-SPR 传感器分析了军团菌的临床样本。该技术已被证明可用于检测那些先前显示 qPCR 定量结果不确定的样本中的突变。当对这些结果进行基准测试时，FO-SPR 结果与 Sanger 测序一致，但与基于荧光的 DNA 解链不一致。所呈现的结果令人信服地支持 FO-SPR 作为一种高分辨率和快速遗传筛选工具的优势，它可以与 SNP 检测的当前标准技术竞争。
