Molecular and Cellular Probes (2001) 15, 81–88 doi:10.1006/mcpr.2000.0343, available online at http://www.idealibrary.com on Detection and quantitation of Aspergillus fumigatus in pure culture using polymerase chain reaction P. Cruz-Perez, M. P. Buttner and L. D. Stetzenbach* Harry Reid Center for Environmental Studies, University of Nevada, Las Vegas 4505 S. Maryland Parkway, Las Vegas, NV 89154-4009, USA (Received 25 October 2000, Accepted 19 December 2000) 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.  2001 Academic Press KEYWORDS: Aspergillus, PCR inhibitors, QPCR, TaqMan TM . INTRODUCTION under-estimated in traditional culture analyses due to its specific growth temperature requirement and the Aspergillus fumigatus is a ubiquitous, thermophilic expertise required in speciating members of the genus Aspergillus. Because numerous fungal spores exist in fungus that grows in damp environments. It is an opportunistic pathogen that affects cystic fibrosis the air and on surfaces both indoors and outdoors, it is advantageous to accurately and rapidly detect patients and the immunocompromised. 1,2 In addition, it is allergenic and has been implicated in several organisms such as A. fumigatus in environmental samples. Due to the detection limitations associated respiratory diseases. 3 Inhalation of Aspergillus spores by susceptible individuals has been linked to the with traditional culture analyses, molecular biology techniques can be utilized for the rapid and sensitive development of pulmonary disease. 4,5 Girardin et al. 6 refer to A. fumigatus as the most common airborne detection of target organisms in indoor environments. The research conducted in this study utilized an fungal pathogen, and Leenders et al. 7 state that A. fumigatus is the predominant Aspergillus species analytical method that has been demonstrated to rapidly and accurately detect airborne virus, 8 bac- involved in invasive infections. However, it is often * Author to whom all correspondence should be addressed at: 4505 S. Maryland Parkway, Las Vegas, NV, 89154-4009; USA. Tel: +1 702 895 1419; Fax: +1 702 895 2688; E-mail: stetzenl@nevada.edu 0890–8508/01/020081+08 $35.00  2001 Academic Press