Real-time PCR for detection of the Aspergillus genus

Abstract

Aspergillus is a genus of mold that has strong indoor sources, including several species capable of acting as opportunistic pathogens. Previous studies suggest that Aspergillus could serve as an indicator for abnormal mold growth or moisture, making it an important genus for environmental monitoring. Here, a quantitative polymerase chain reaction (qPCR, or real-time PCR) assay is presented for Aspergillus. The assay shows good specificity for the genus, detecting all Aspergillus species tested, although a few non-Aspergillus species are also amplified. Sensitivity testing demonstrates that DNA representing one conidium can be detected. A validation study compared qPCR results against direct microscopy counts using A. fumigatus conidia aerosolized into a laboratory chamber. The assay was then used to quantify Aspergillus in indoor air samples, demonstrating its utility for environmental monitoring. Analysis of a small number of clinical sputum samples showed complete agreement with culturing results.

Contributions of Foot Traffic and Outdoor Concentrations to Indoor AirborneAspergillus

“…Each qPCR run included a standard curve, with R 2 ≥ 0.95, and slope of -3.81 ± 0.17. This corresponded to the slope of the calibration curve and its standard error found using direct microscopy (Goebes et al 2007). Results were calibrated using standard curves made with DNA extracted from A. fumigatus conidia.…”

“…A full discussion of the specificity and sensitivity of the assay is presented in Goebes et al (2007). In brief, the assay strongly detected all tested Aspergillus species.…”

“…The entire methodology for DNA extraction, purification and qPCR, as well as the validation of the qPCR assay, is presented in Goebes et al (2007). A summary and major details are presented here.…”

Total viable molds and fungal DNA in classrooms and association with respiratory health and pulmonary function of European schoolchildren

“…It could be that, among the children undergoing spirometry, those of Reims, besides being younger, had also lower mean weight and the equations of Quanier, not including weight, might excessively overestimate the spirometry of Reims. Body mass index seems to affect lung function in children quite differently from adults and there is evidence that FEV 1 and FVC increase proportionally in relation to an increase in BMI (29). Recently, it was found that the measured FEV 1 and FVC values, in normal weight and underweight children, were lower than the values predicted via equations not including weight; in contrast, the values predicted via the equations including the body weight were closer to measured values (29).…”

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under MixedEimeriaInfection

“…The development of molecular techniques has streamlined fungal detection and quantification. Communal DNA or molecular probes can be used to target species as well as to facilitate the monitoring of entire fungal communities (Gharizadeh et al, 2004;Goebes et al, 2007;Griffiths et al, 2006;May et al, 2001;Scupham et al, 2006;Urubschurov et al, 2008). Scupham et al (2006) estimated that fungi comprise 0-10%, with a median of 2%, of biota associated with murine cecal biofilm, and that fungi sparsely populated digesta outside the ceca.…”