Specific detection of Stachybotrys chartarum in pure culture using quantitative polymerase chain reaction

Abstract

Research was conducted with laboratory cultures to establish a protocol for the rapid detection and quantitation of the toxigenic fungus Stachybotrys chartarum by means of polymerase chain reaction (PCR). Sequences for the 18 S rRNA gene of S. chartarum were obtained from GenBank and compared against all other available sequences on-line with the Basic Local Alignment Search Tool (BLAST). Two sets of TaqMan primers and one fluorescently labelled probe were designed and tested for selectivity, specificity and sensitivity of detection. A fluorogenic nuclease assay in conjunction with a sequence detector were used for the amplification and quantitation of S. chartarum. The primers designed amplified all S. chartarum isolates tested and did not amplify DNA extracted from other Stachybotrys species or 15 other fungal genera. The primer set selected had a sensitivity of <23 template copies. Many S. chartarum samples were initially negative after PCR amplification. Incorporation of an internal positive control in the PCR reaction demonstrated the presence of inhibitors in these samples. PCR inhibitors were removed by dilution or further purification of the DNA samples. The results of this research report on a quantitative PCR (QPCR) method for detection and quantitation of S. chartarum and demonstrate the presence of PCR inhibitors in some S. chartarum isolates.