Construction of internal control for the quantitative assay of Aspergillus fumigatus using real-time nucleic acid sequence-based amplification.
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 10(4) to 10(8) copies/mL of RNA and 10(0) to 10(8) 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.
Yoo
Jin-Hong
JH
Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
Choi
Su-Mi
SM
Choi
Jung-Hyun
JH
Kwon
Eun-Young
EY
Park
Chulmin
C
Shin
Wan-Shik
WS
D000192:Actinomycetales / Q000235:genetics; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D017123:Bacteriophage T7 / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D012015:Reference Standards; D021161:Self-Sustained Sequence Replication / Q000592:standards*