Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples

James J. McDevitt, Peter Sj Lees, William G. Merz, Kellogg Schwab

Research output: Contribution to journalArticle

Abstract

Exposure to Aspergillus fumigatus is linked with respiratory diseases such as asthma, invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis. Molecular methods using quantitative PCR (qPCR) offer advantages over culture and optical methods for estimating human exposures to microbiological agents such as fungi. We describe an assay that uses lyticase to digest A. fumigatus conidia followed by TaqMan™ qPCR to quantify released DNA. This method will allow analysis of airborne A. fumigatus samples collected over extended time periods and provide a more representative assessment of chronic exposure. The method was optimized for environmental samples and incorporates: single tube sample preparation to reduce sample loss, maintain simplicity, and avoid contamination; hot start amplification to reduce non-specific primer/probe annealing; and uracil-N-glycosylase to prevent carryover contamination. An A. fumigatus internal standard was developed and used to detect PCR inhibitors potentially found in air samples. The assay detected fewer than 10 A. fumigatus conidia per qPCR reaction and quantified conidia over a 4-log10 range with high linearity (R2 > 0.99) and low variability among replicate standards (CV = 2.0%) in less than 4 h. The sensitivity and linearity of qPCR for conidia deposited on filters was equivalent to conidia calibration standards. A. fumigatus DNA from 8 isolates was consistently quantified using this method, while non-specific DNA from 14 common environmental fungi, including 6 other Aspergillus species, was not detected. This method provides a means of analyzing long term air samples collected on filters which may enable investigators to correlate airborne environmental A. fumigatus conidia concentrations with adverse health effects.

Original languageEnglish (US)
Pages (from-to)325-335
Number of pages11
JournalMycopathologia
Volume158
Issue number3
DOIs
StatePublished - Oct 2004

Fingerprint

Aspergillus fumigatus
Fungal Spores
conidia
quantitative polymerase chain reaction
Air
air
Polymerase Chain Reaction
aspergillosis
sampling
methodology
DNA
Fungi
Uracil-DNA Glycosidase
Allergic Bronchopulmonary Aspergillosis
glycosylases
Extrinsic Allergic Alveolitis
uracil
fungi
Aspergillosis
annealing

Keywords

  • Air monitoring
  • Airborne fungi
  • Aspergillus fumigatus
  • Conidia
  • Filter
  • Quantitative PCR
  • Sample inhibition

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Plant Science

Cite this

Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples. / McDevitt, James J.; Lees, Peter Sj; Merz, William G.; Schwab, Kellogg.

In: Mycopathologia, Vol. 158, No. 3, 10.2004, p. 325-335.

Research output: Contribution to journalArticle

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