Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan(TM)fluorogenic probe system.
The occurrence of Stachybotrys chartarum in indoor environments has been associated with a number of human health concerns, including fatal pulmonary haemosiderosis in infants. Currently used culture-based and microscopic methods of fungal species identification are poorly suited to providing quick and accurate estimates of airborne human exposures to the toxin containing conidia of this organism. In this study, real-time polymerase chain reaction (PCR) product analysis using the TaqManU fluorogenic probe system and an Applied Biosystems PrismS model 7700 sequence detection instrument (model 7700) was applied to the specific detection of S. chartarum ribosomal DNA (rDNA) sequences. Based upon this assay and a recently reported comparative cycle threshold method for quantifying target DNA sequences using data from the model 7700, a simple method for the direct quantification of S. chartarum conidia was developed. In analyses of samples containing several different strains and from two to over 2x10(5)cells, this method consistently provided quantitative estimates of S. chartarum conidia that were within a one-fold range (50-200%) of those determined on the basis of direct microscopic counts in a haemocytometer. The method showed a similar level of agreement with direct counting in the quantification of S. chartarum conidia in air samples collected from several contaminated homes.
Haugland
R A
RA
National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio, 45268, USA.haugland.rich@epa.gov
Vesper
S J
SJ
Wymer
L J
LJ
D016902:Air Pollution, Indoor / Q000032:analysis; D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis; D004783:Environmental Microbiology; D005456:Fluorescent Dyes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013191:Stachybotrys / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005456:Fluorescent Dyes