title: Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.
abstract: Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.
authors.affiliation: Division of Molecular Microbiology, Children's Cancer Research Institute (CCRI), Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Czurda
authors.affiliation: Division of Molecular Microbiology, Children's Cancer Research Institute (CCRI), Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Smelik
authors.affiliation: Division of Molecular Microbiology, Children's Cancer Research Institute (CCRI), Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Preuner-Stix
authors.affiliation: Division of Molecular Microbiology, Children's Cancer Research Institute (CCRI), Vienna, Austria.
authors.forename: F
authors.initials: F
authors.lastname: Nogueira
authors.affiliation: Division of Molecular Microbiology, Children's Cancer Research Institute (CCRI), Vienna, Austria thomas.lion@ccri.at.
authors.forename: T
authors.initials: T
authors.lastname: Lion
mesh_terms: D004271:DNA, Fungal / Q000302:isolation & purification* / Q000378:metabolism; D003666:Decontamination / Q000379:methods*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005189:False Positive Reactions; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D009181:Mycoses / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004271:DNA, Fungal; D007202:Indicators and Reagents; D003851:Deoxyribonucleases