Validation of Automated DNA Extraction Methods of Investigational Quantitative PCR for Detection of Invasive Pulmonary Aspergillosis

T. J. Walsh, M. Kasai, G. A. Fahle, R. Petraitiene1, V. Petraitis1,3, C. Beveridge1, H. Wubneh, J. D. Bacher, P. R. Murray

Author address: 

NCI, Bethesda, MD, Dept of Lab. Med., NIH CC, Bethesda, MD, LASP, SAIC-Frederick, Inc., Frederick, MD, DVR, ORS, NIH, Bethesda, MD.

Abstract: 

Background: Quantitative PCR (qPCR) and other molecular diagnostic methods may improve detection of pathogenic Aspergillus spp. in patients with invasive pulmonary aspergillosis (IPA). Extraction of DNA from fungal cells is a critical method in the development, optimization and application of qPCR. In translating qPCR assays from our investigational laboratory to the Department of Laboratory Medicine (DLM) for detection of Aspergillus spp. in bronchoalveolar lavage (BAL) and tissue, we sought to standardize and validate two major automated DNA extraction methodologies. Methods: We initially developed our investigational qPCR system for detection of experimental IPA by using comparable manual DNA extraction methods and an automated method (MagNA Pure (MNP (Roche)). We then hypothesized that the qPCR assays performed using the automated DNA extraction platform of EasyMag (EM (bioMí©rieux)) by the DLM would yield results comparable to that using the MNP. Results: Similar amounts of mean log DNA/ml (±SEM) were extracted from 100 μl supernatants of RPMI media containing A. fumigatus germinated conidia by MNP (5.50±0.18) and EM with Roche lysis buffer (LB) (4.90±0.25), EM+LB+proteinase K (4.89±0.19), and EM+LB (4.83±0.21). Yield from DNA kinetic studies of RPMI media containing A. fumigatus germinated conidia measured over 0, 2, 4 and 6h also revealed similar mean log DNA/ml yields for MNP (4.56±0.20 - 5.63±0.12) and EM+LB (3.32±0.68 - 4.78±0.11). Comparable mean log DNA yields for 10-fold serial dilution of A. fumigatus genomic DNA (1x10^6 fg/100μl - 1x10^0 fg/100μl) were obtained for both systems: MNP (0-5.22±0.06) and EM+LB (2.39±0.30-5.74±0.13). Mean log DNA/ml yields from lung tissue and BAL from the neutropenic rabbit model of IPA also were similar: lung tissue [MNP (3.41±1.13) vs EM+LB (3.91±0.94), (95% CI 0.50, 6.31 vs 1.50, 6.31)] and BAL [MNP (2.66±0.99) vs EM+LB (2.70±1.04), (95% CI -0.48, 5.81 vs -0.61, 6.02)]. Conclusions: We conclude that MNP and EM+LB yield comparable amounts of genomic DNA from A. fumigatus in different matrices and are each suitable for qPCR assays in detection of IPA
2010

abstract No: 

F-2146

Full conference title: 

110th General Meeting American Society for Microbiology
    • ASM 110th (2010)