A novel clinical diagnostic assay for the identification of multiple human fungal pathogens in a single sample
Abstract number: P2096
Dempsey K., Ricketts A., Fitchett K., Redden J., White P., Perry M., Barnes R.
Objectives: Surface-Enhanced Resonance Raman Scattering (SERRS) is a highly sensitive and molecular-specific detection system which can be used to detect multiple human pathogens in a single sample. The objective of this study was to develop a screening assay for the multiplex detection of fungal targets using PCR and SERRS.
Methods: The assay was designed to detect 23 species of fungi known to cause invasive fungal disease (IFD). As Candida and Aspergillus species account for >80% of all IFDs, one well of this assay was specifically designed to detect a broad range of both genera and specifically identified C. glabrata and C. krusei as these organisms may require alternative antifungal therapy. A second well of this assay is being developed to detect other causes of IFD.
A multiplex diagnostic assay was developed to detect targets at low copy number in clinically relevant samples. Universal primers and specific probes were designed to amplify and detect each target. One primer from each primer set was biotinylated to allow capture of the amplified DNA by streptavidin-coated beads. A labelled probe sequence specific for each target was hybridised to the PCR products and captured on the beads. The DNA/probe complex was washed, the probe released then analysed by SERRS.
Results: Multiplex PCR for both genera amplified fungal species from a single sample. Each was successfully identified by SERRS using the probe's unique molecular fingerprint, confirming the presence or absence of each target.
The function of this multiplex assay is as diagnostic screening test to exclude the presence of invasive fungal disease and requires excellent analytical sensitivity. The analytical thresholds for the detection of Candida and Aspergillus species are equivalent to real-time PCR assays. The reproducible 100% detection limit for the targets of this assay is 20 input copies, which correlates to less than 1 genome per reaction (Table 1).
Conclusions: This clinically relevant assay demonstrates excellent sensitivity and specificity for the range of fungal species tested. The reliably detection of common fungal species at 20 copies was clearly demonstrated.
|Session name:||Abstracts of 21st ECCMID / 27th ICC|
|Location:||Milan, Italy, 7 - 10 May 2011|
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