Evaluation of nucleic acid sequence based amplification using fluorescence resonance energy transfer (FRET-NASBA) in quantitative detection of Aspergillus 18S rRNA.
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.
Park
Chulmin
C
Catholic Research Institutes of Medical Science, The Catholic University of Korea, College of Medicine, Seoul, South Korea.
Kwon
Eun-Young
EY
Shin
Na-Young
NY
Choi
Su-Mi
SM
Kim
Si-Hyun
SH
Park
Sun Hee
SH
Lee
Dong-Gun
DG
Choi
Jung-Hyun
JH
Yoo
Jin-Hong
JH
D001228:Aspergillosis / Q000175:diagnosis*; D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D009172:Mycology / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S