Spherical Nucleic Acid Enhanced FO-SPR DNA Melting for Detection of Mutations in Legionella pneumophila

• Published in: Analytical chemistry (Washington) 2013-02, Vol.85 (3), p.1734-1742
• Main Authors: Knez, Karel, Janssen, Kris P. F, Spasic, Dragana, Declerck, Priscilla, Vanysacker, Louise, Denis, Carla, Tran, Dinh T, Lammertyn, Jeroen
• Format: Article
• Language: English
• Subjects: Base Sequence; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Fiber Optic Technology - methods; Fluorescence; Gram-negative bacteria; Legionella pneumophila - genetics; Legionella pneumophila - isolation & purification; Melting; Molecular Sequence Data; Mutation; Mutation - genetics; Nanoparticles; Nucleic Acid Denaturation - genetics; Nucleic Acids - genetics; Nucleic Acids - isolation & purification; Surface Plasmon Resonance - methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac303008f

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.