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Obtaining DNA template of adequate purity for amplification and avoiding compounds present that inhibit amplification: A Verticillium dahliae example
Guillaume J. Bilodeau: Canadian Food Inspection Agency
<div><span>Obtaining a good quality and yield from a DNA extraction is the key in direct detection samples for looking at plant pathogens and organism. In best practices in diagnostic test development, the quality of the DNA is the first step to optimize in order to have success. Over the years, in different projects, we evaluated different DNA extraction methods with optimization in order to obtain the best DNA for Real-Time PCR detection and some metagenomics experiments. Real-time PCR was employed as a tool to determine the best extraction method. Internal controls and specific qPCR assays for <i>Verticillium</i><i> dahliae</i> and other target organisms were used to determine PCR inhibition (quality) and DNA yield. Soil and water samples were collected and spiked with the target organisms to evaluate the best DNA extraction procedure. From the DNA extraction methods tested, some additional steps with magnetic bead purification and chemical flocculation to remove PCR inhibitors were evaluated. Clearly, the methods giving the highest DNA concentration do not always provide the best quality of DNA, and the ones with the best quality do not provide the highest yield. Removal of PCR inhibitors such as humic acids usually requires addition of a chemical flocculant during the extraction, or a post-extraction step such as magnetic bead-based purification. Further using qPCR to directly targeting organisms, some comparison of kits and methods have also been done for metagenomic analysis using Next generation sequencing (NGS).</span></div>

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