Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at ≥1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was ≈70 pg/μl or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.
- SYBR green I
- Spore mats