Evaluation of nucleic acid sequence based amplification using fluorescence resonance energy transfer (FRET-NASBA) in quantitative detection of Aspergillus 18S rRNA

Catholic Research Institutes of Medical Science, The Catholic University of Korea, College of Medicine, Seoul, South Korea.
Medical mycology: official publication of the International Society for Human and Animal Mycology (Impact Factor: 2.34). 01/2011; 49(1):73-9. DOI: 10.3109/13693786.2010.507604
Source: PubMed


We attempted to apply fluorescence resonance energy transfer technology to nucleic acid sequence-based amplification (FRET-NASBA) on the platform of the LightCycler system to detect Aspergillus species. Primers and probes for the Aspergillus 18S rRNA were newly designed to avoid overlapping with homologous sequences of human 18s rRNA. NASBA using molecular beacon (MB) showed non-specific results which have been frequently observed from controls, although it showed higher sensitivity (10(-2) amol) than the FRET. FRET-NASBA showed a sensitivity of 10(-1) amol and a high fidelity of reproducibility from controls. As FRET technology was successfully applied to the NASBA assay, it could contribute to diverse development of the NASBA assay. These results suggest that FRET-NASBA could replace previous NASBA techniques in the detection of Aspergillus.

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    • "At present, a number of different variations of real-time PCR procedures are available, allowing the quantification of fungal load and, depending on the setting of the assay, pathogen identification at the species level (Basková et al. 2007; Schabereiter- Gurtner et al. 2007). Moreover, special methods such as nucleic acid sequence-based amplification (Park et al. 2011), multiplex PCR followed by DNA microarray (Spiess et al. 2007), fragment size analysis of variable regions of the fungal genome (Landlinger et al. 2009a), DNA sequencing (Leaw et al. 2006), hybridization to specific capture probes bound to microbeads (Landlinger et al. 2009b), or pyrosequencing (Borman et al. 2010) have been developed to identify fungal pathogens in clinical samples. "
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