Detection and quantitation of Aspergillus fumigatus in pure culture using polymerase chain reaction.
Research was conducted with laboratory cultures to establish a protocol for the rapid detection and quantitation of the thermophilic fungus, Aspergillus fumigatus, using genetic amplification. Oligonucleotide primers and a fluorescently labelled probe were designed for use with quantitative polymerase chain reaction (QPCR). Primers and probe were tested for selectivity, specificity and sensitivity of detection of the target organism using a fluorogenic nuclease assay and a sequence detector. The DNA extraction protocol consisted of enzymatic treatment and boiling of fungal spore suspensions followed by DNA concentration and purification. The primer set developed was specific for A. fumigatus and had a sensitivity of <20 template copies. These primers amplified all A. fumigatus isolates tested and did not amplify DNA extracted from other Aspergillus species or 15 other fungal genera. However, one A. fumigatus sample was initially negative after PCR amplification. Incorporation of an internal positive control in the PCR reaction demonstrated the presence of inhibitors in this and other samples. PCR inhibitors were removed by dilution or further purification of the DNA samples. This research resulted in a QPCR method for detection and quantitation of A. fumigatus and demonstrated the presence of PCR inhibitors in several A. fumigatus isolates.
Cruz-Perez
P
P
Harry Reid Center for Environmental Studies, University of Nevada, Las Vegas 4505 S. Maryland Parkway, Las Vegas, NV 89154-4009, USA.
Buttner
M P
MP
Stetzenbach
L D
LD
D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal / Q000302:isolation & purification; D005456:Fluorescent Dyes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D012335:RNA, Ribosomal