Development of a Real-Time Polymerase Chain Reaction (PCR) Assay using Hybridisation Probes to Detect the Fungus Aspergillus

We developed a real-time PCR assay on the LightCycler instrument (Roche Molecular Systems) to detect the presence of Aspergillus, by detecting a 164bp fragment of the 18S ribosomal DNA (rDNA) gene. rDNA has traditionally been used as a phylogenetic marker in determining the relationship of one species or genus to another. Molecular identification of the 164bp amplicon was carried out using a pair of hybridisation probes, which rely on fluorescence resonance energy transfer to detect minute quantities of 18S rDNA. The assay was highly accurate (100 % sensitivity; 100 % specificity) in detecting various Aspergillus species in culture and five out of five samples containing 100ƒg of genomic DNA (corresponding to ~200 copies of the 18S rDNA gene in the starting material). It can in fact detect down to 10ƒg of DNA, with 60-80 % consistency. There was no cross-reactivity with all 6 clinical Candida isolates tested. It achieved relatively less success (50 % detection rate, n=6) when applied to detection of Aspergillus in paraffin-embedded tissues showing the characteristic morphologic appearance of septate, branched hyphae, used to identify fungi in tissue sections by histopathologists. This prototype assay has the potential to be a rapid (1h amplification and detection time), accurate and highly specific assay, which can be adapted for diagnosis and monitoring of invasiv