MYCASSAY ASPERGILLUS: A CE-MARKED REAL-TIME PCR ASSAY FOR DETECTING ASPERGILLUS DNA IN RESPIRATORY SAMPLES

S. Follett1, R. O'Neill1, A. Moody1*

Author address: 

1Myconostica

Abstract: 

Purpose: Routine diagnosis of invasive pulmonary aspergillosis includes microscopy and culture from bronchoalveolar lavage (BAL), as well as Aspergillus antigen testing of blood. These methods have known limitations, and the use of a molecular diagnostic approach has the potential to enhance patient outcome. While the utility of molecular diagnostics has long been recognised, the widespread application has been restricted by the lack of a standardised, commercially validated assay. This work describes the analytical validation and initial clinical assessment of a molecular diagnostic kit for the detection of Aspergillus spp. genomic DNA using molecular beacon PCR technology. The whole test procedure, including extraction of DNA from the clinical sample (using the CE-marked MycXtra® Fungal DNA Extraction kit), can be completed within 4 hours. Methods: Oligonucleotide primers and molecular beacon were designed to specifically target a section of the Aspergillus ribosomal 18S gene. Also included in the reaction mixture is an Internal Amplification Control (IAC) system incorporating, a DNA fragment not present in Aspergilli, other fungal, bacterial or human genomes. The analytical sensitivity, selectivity and specificity were determined along with an assessment of clinically relevant interfering substances. Clinical BAL samples were extracted using the MycXtraTM Fungal DNA Extraction kit and the performance assessed. A clinical cut-off (CCO) was established to differentiate between disease and non-disease states at low levels of Aspergillus DNA. Results: The assay has an analytical limit of detection of
2010

abstract No: 

117

Full conference title: 

4th Advances Against Aspergillosis
    • AAA 4th (2010)