Background: Real-time quantitative PCR (qPCR) has become the most widely used molecular
technology for diagnostic applications designed to detect and quantify pathogens. Aspergillus-specific
qPCR assays have been proposed as alternatives to conventional diagnostic procedures for Invasive
Aspergillosis, where early diagnosis and treatment are critical. Our objective was to develop a qPCR
kit designed to detect clinically relevant Aspergillus species whilst simultaneously capable of
identifying Aspergillus terreus, an amphotericin-resistant fungus associated with a high mortality rate.
Material/methods: Assay design, optimisation and validation were performed in strict compliance with
the MIQE guidelines (1). Sequences of target organisms were aligned in CLC Sequence Viewer to
identify suitable target sequences. Primer and probe sequences were designed by Beacon Designer.
In silico analysis was performed using nucleotide BLAST and the target secondary structure/template
accessibility was assessed using MFOLD. SYBR Green chemistry and melt curve analysis were used
to determine optimal annealing temperatures and optimal primer/probe concentrations. Pan-
Aspergillus (FAM) and A. terreus-specific (HEX) hydrolysis probe assays were combined with our own
internal extraction control assay (ROX) and optimal conditions were established to create AspID. The
assay was extensively validated using DNA extracts from fungal cultures, clinical bronchial washes
and serum samples, AsTeC Consortium Aspergillus calibrator material (2) and the EAPCRI DNA panel
Results: Under optimal PCR conditions the primers in OLM’s AspID kits result in amplification
efficiencies of >90%. The test has a broad dynamic range of at least six orders of magnitude and can
detect down to around 10 copies of target template. Since AspID targets a region of DNA that has
numerous repeats, this is the equivalent of less than one fungal genome. AspID detects all targets
designated ‘Essential’ in the EAPCRI 2013 panel.
Conclusions: The AspID multiplex qPCR test kit sensitively and specifically detects genomic DNA of clinically relevant Aspergillus species, with simultaneous identification of Aspergillus terreus, which provides clinically useful information due to the latter’s intrinsic resistance to Amphotericin B.
Full conference title:
- ECCMID 27th (2017)