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 falsepositive results in highly sensitive molecular fungal detection assays. N ucleic acid amplification-based techniques can detect minute quantities of fungal DNA and can enable fast and sensitive screening in patients at risk for invasive fungal infections (IFI). They can overcome the limitations of conventional diagnostic methods that are slow or have inadequate sensitivities (1-3). However, a major drawback of highly sensitive PCR applications is that even low-level contaminations can lead to false-positive results (4). This is particularly evident when broad-range screening methods are applied because their ability to detect contaminating DNA from many different fungal species renders them more vulnerable to trace amounts of nucleic acid contamination than species-specific assays (1). This problem is already well described for universal bacterial assays, where PCR testing based on 16S rRNA gene amplification can be compromised by DNA contamination of reagents derived from bacterial sources such as Taq DNA polymerase or uracil-N-glycosylase (5, 6).Problems with fungal contamination during PCR-based assays have been previously reported. Airborne conidia or spores and trace amounts of fungal nucleic acids in commercially available enzymes used for fungal cell lysis and in components of DNA extraction kits were identified as possible sources of contamination (7-10). Here we provide the first evidence for the presence of fungal DNA contaminants in lyophilized primers and TaqMan probes as well as in master mix solutions for real-time PCR analysis purchased at renowned biotechnology companies. Using a TaqMan-based panfungal PCR assay (11) establi...

The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

“…The amplification of background contaminants from PCR reagents could perhaps be avoided via the use of primer-extension PCR ( 102 ), but this would have no effect on contamination originating from other sources. Several methods have been suggested to remove contaminating DNA from reagents and the lab environment, including UV and gamma radiation ( 103 – 107 ); DNA intercalation by 8-methoxypsoralen, ethidium monoazide, and propidium monoazide ( 104 , 106 – 108 ); enzymatic treatments ( 105 – 107 , 109 – 111 ); silica-based membrane filtration ( 112 ); CsCl 2 density gradient centrifugation ( 111 ); and bleach/copper-bis-(phenanthroline)-sulfate/H 2 O 2 (CoPA) solution treatment ( 105 ). These methods have shown varied effects on contamination levels and PCR sensitivity, and the inclusion of reagent-only controls alongside these decontamination measures is still recommended.…”

Viral Surrogates in Potable Reuse Applications: Evaluation of a Membrane Bioreactor and Full Advanced Treatment

“…The more conservative USEPA approach was used for the 16S rRNA gene assay, while the alternative approach was used for the remaining molecular targets. The justification for use of the higher LoQ for the 16S rRNA gene was to mitigate the effects of false amplification, which can be common for 16S rRNA gene assays due to inherent DNA contamination of master mixes (Czurda et al 2015;Mühl et al 2010;Rand and Houck 1990;Rueckert and Morgan 2007;Salter et al 2014;Spangler et al 2009;Tondeur et al 2004). The assay-specific LoQs are summarized in Table S4 LoQ ¼ ts ð1Þ…”

Monitoring Fungal Communities With the Global Spore Sampling Project

“…The abundance of Cladosporium species at all sampling locations raises the possibility of contamination (Czurda et al, 2016). What speaks against this is that the total amount of DNA varied by five orders of magnitude among the samples, while the proportion of Cladosporium remained relatively stable.…”