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United States Department of Agriculture

Agricultural Research Service

Title: A RAPID METHOD FOR DETECTION AND QUANTIFICATION OF BACTERIAL DNA IN RUST FUNGAL DNA SAMPLES

Authors
item Barnes, Charles
item Szabo, Les

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 7, 2007
Publication Date: January 15, 2008
Citation: Barnes, C.W., Szabo, L.J. 2008. A Rapid Method for Detection and Quantification of Bacterial DNA in Rust Fungal DNA Samples. Phytopathology. 98:115-119.

Interpretive Summary: Rust fungi are obligate parasitic plant pathogens and molecular analysis often depends on extraction of DNA from asexual urediniospores collected from plant tissue. Bacterial DNA contamination of the rust fungal DNA can be a significant problem. To determine bacterial DNA contamination levels, a quantitative real-time PCR (Q-PCR) assay was developed. DNA primers previously described by Schwieger and Tebbe (1998), which are able to amplify a segment of nuclear ribosomal rDNA from a diverse group of bacteria, was used in the development of this assay. Verification of the accuracy of this assay was demonstrated by adding known quantities of bacterial DNA to samples of fungal DNA. The assay was shown to accurately detect levels of bacterial contamination from 0.1% to over 10%. Levels of bacterial contamination were determined for DNA samples from rust fungi (P. graminis, P. coronata, P. striiformis, and P. triticina), which ranged from 0.1% to 10%. At low levels of bacterial DNA contamination (less than 1%) amplification from the fungal DNA occurred. This problem was easily resolved by adding 100-200 ng of bacterial DNA to the samples. Scientists working on molecular analysis of rust fungi, as well as, scientists working with other systems will use this information.

Technical Abstract: Rust fungi are obligate parasitic plant pathogens and molecular analysis often depends on extraction of DNA from asexual urediniospores collected from plant tissue. Bacterial DNA contamination of the rust fungal DNA can be a significant problem. A quantitative real-time polymerase chain reaction (Q-PCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples based on universal bacterial primers Com1/Com2 (Schwieger and Tebbe, 1998). Using Escherichia coli DNA as a standard, the assay was linear over the range of 0.02 - 20 ng. The accuracy of the assay was verified by adding known quantities of E. coli DNA to Puccinia graminis DNA. DNA samples from P. graminis, P. coronata, P. striiformis and P. triticina was tested and shown to have levels of bacterial contamination ranging from 0.1 ' 10%. However, when bacterial DNA concentration was less than 1.0%, two amplicons were often produced as a result of amplification from fungal DNA. Spiking the fungal DNA samples with 100 ' 200 pg of E. coli DNA successfully eliminated the amplification of fungal DNA, allowing the quantitation of contaminating bacterial DNA to a level of approximately 0.1%. This assays provides a rapid method to quantify levels of contaminating bacterial in DNA samples.

Last Modified: 9/1/2014