Contaminations occurring in fungal PCR assays

Abstract

Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken.

FIG. 1

Gel electrophoresis (2% TAE-agarose gel) showing amplified DNA of potentially contaminated samples (specific bands of approximately 500-bp length). Lanes: 1, 100-bp marker; 2, zymolyase, contaminated lot; 3, zymolyase, contaminated lot, 1:10 dilution; 4, 10× buffer from digoxigenin (Dig)-labeling kit (Boehringer Mannheim), contaminated lot; 5, 10× buffer from Dig-labeling kit (Boehringer Mannheim), contaminated lot; 6, zymolyase, regular lot, not contaminated; 7, proteinase K, regular lot, not contaminated; 8, lyticase, contaminated lot; 9, lysing enzyme extracted from T. harzianum; 10, positive control, A. fumigatus DNA, extracted from 10³ CFU; 11, negative control of the extraction; and 12, negative control of the PCR.