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.

Download full-text


Available from: Si-Hyun Kim
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Although invasive fungal diseases (IFDs) are relatively rare, they have become an increasingly common life-threatening complication in a variety of critically ill patients. Due to changes in treatment strategies, patterns of IFDs have changed substantially as well. Yeast infections have shifted toward a higher proportion of non-albicans Candida species, but their overall incidence has remained stable. In contrast, IFDs caused by molds, including particularly various species of Aspergillus, Fusarium, and Mucorales, have increased in number. In view of the growing incidence and the high mortality rates of IFDs, accurate diagnostic techniques permitting timely onset of adequate antifungal treatment are of paramount importance. Although conventional approaches such as microscopy, cultivation, histopathological examination, and imaging methods still represent the gold standard, the diagnosis remains difficult because of limited sensitivity and specificity. Noninvasive and culture-independent diagnostic techniques, including fungal antigen detection, and different molecular-based techniques are becoming increasingly important. Of the fungal surrogate markers such as cell wall components, galactomannan and (1,3)-β-D-glucan by commercially available diagnostic kits have become widely used, but the results are still controversial. A plethora of PCR-based diagnostic methods targeting different gene regions and exploiting a variety of amplicon detection tools have been published. Molecular assays have the capacity to overcome the limitations of other diagnostic approaches, but the current lack of methodological standardization and validation, together with not always clear interpretation of the results, has prevented broad application in the clinical setting.
    Full-text · Article · May 2012 · Folia Microbiologica
  • Source
    • "We have devised real-time NASBA (RTi-NASBA) using molecular beacon and applied this technique for the diagnosis of IA in patients with febrile neutropenia (10-12). Recently, we have tried a new paradigm of RTi-NASBA on the platform of the LightCycler 480 system (13). However, there has been little knowledge about the practical usefulness of NASBA for evaluation of the treatment response to antifungal therapy in patients suffering from IA. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Monitoring the response to therapy for invasive aspergillosis (IA) is essential for the management of patients with hematologic diseases. We evaluated the correlation between the outcome of real-time nucleic acid sequence-based amplification (RTi-NASBA) for Aspergillus 18S rRNA and the clinical outcome of IA. A total of 157 serum samples from 29 patients with IA were tested for RTi-NASBA. The treatment response and mortality were compared with the NASBA outcome (whether the NASBA value was converted to negative or not) at 12 weeks after the start of antifungal therapy. At 12 weeks, there was a moderate correlation between the treatment failure and persistently positive NASBA (κ = 0.482; P = 0.019). Deaths attributable to IA were more prevalent in patients without negative conversion of NASBA than in those with negative conversion (50% vs 5%; P = 0.013). Significant factors of treatment failure at 12 weeks were the status of hematologic disease (nonremission; P = 0.041) and the NASBA outcome (failure of negative conversion; P = 0.024). Survival was significantly better in patients with negative conversion of NASBA than those with persistently positive values (P = 0.036). This study suggests that the serial monitoring of RTi-NASBA could be useful for prediction of the clinical outcome in hematologic patients with IA.
    Full-text · Article · Jan 2012 · Journal of Korean medical science
  • Source

    Full-text · Dataset ·
Show more