Construction of internal control for the quantitative assay of Aspergillus fumigatus using real-time nucleic acid sequence-based amplification

Abstract We have developed a scheme for quantitative real-time (RTi) nucleic acid sequence-based amplification (NASBA) for the detection of Aspergillus fumigatus using internal control (IC) RNA. Construction of IC RNA began with the synthesis of nontarget sequences from Clavibacter michiganensis sub...

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Bibliographic Details
Published in:Diagnostic microbiology and infectious disease 2008, Vol.60 (1), p.121-124
Main Authors: Yoo, Jin-Hong, Choi, Su-Mi, Choi, Jung-Hyun, Kwon, Eun-Young, Park, Chulmin, Shin, Wan-Shik
Format: Article
Language:English
Subjects:
Actinomycetales - genetics
Aspergillosis - diagnosis
Aspergillus
Aspergillus fumigatus
Aspergillus fumigatus - genetics
Aspergillus fumigatus - isolation & purification
Bacteriophage T7 - genetics
Biological and medical sciences
Clavibacter michiganensis
Fundamental and applied biological sciences. Psychology
Humans
Infectious Disease
Internal control
Internal Medicine
Microbiology
Miscellaneous
Mycology
NASBA
Promoter Regions, Genetic
Reference Standards
Self-Sustained Sequence Replication - standards
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Summary:Abstract We have developed a scheme for quantitative real-time (RTi) nucleic acid sequence-based amplification (NASBA) for the detection of Aspergillus fumigatus using internal control (IC) RNA. Construction of IC RNA began with the synthesis of nontarget sequences from Clavibacter michiganensis subsp. sepedonicus by a primary polymerase chain reaction (PCR) step, followed by a secondary PCR step using chimeric primers to produce a chimeric sequence including a T7 promoter region. Finally, chimeric IC RNAs were constructed by the use of in vitro transcription. The assay detected A. fumigatus within a range of 104 to 108 copies/mL of RNA and 100 to 108 cells. When the assay was performed with the target and IC RNA in 1 reaction in a single tube, there was little interference of the IC RNA in the measurement of the amount of target. The amount of RNA calculated using the assay was not significantly different from the amount of input RNA as indicated by Bland–Altman analysis. The IC RNA we constructed can be used in RTi-NASBA for quantitative detection of Aspergillus with good precision and accuracy.
ISSN:0732-8893
1879-0070
DOI:10.1016/j.diagmicrobio.2007.08.001