Improved DNA extraction method for Verticillium detection and quantification in large-scale studies using PCR-based techniques

Abstract

An improved plant and soil DNA extraction method for detection and quantification of Verticillium species using polymerase chain reaction (PCR) based techniques is presented. This method involves the use of extraction buffer containing proteinase K and further DNA purification with addition of ammonium acetate. In the case of soils, the protocol combines the benefits of using a commonly reported nucleic acid carrier with the simplicity of the proteinase K - ammonium acetate method. As organic solvent extractions are not needed and the DNA extractions can be performed in small volumes, this method becomes a very attractive alternative when a large number of plant tissue or soil samples have to be processed for PCR detection and (or) quantification. In addition, comparative studies with the traditional SDS buffer - phenol protocol showed that the general level of PCR inhibition is reduced, especially in soil samples, when the proteinase K - ammonium acetate method is used. Inhibition is one of the more serious limitations of PCR applied to quantitative studies. Thus the use of this simple DNA extraction method, which is also effective in reducing the level of PCR inhibitory factors, represents an improved alternative for detection and quantification of Verticillium spp., in both plant tissue and soil samples.

Keywords:

• Verticillium detection
• PCR
• DNA extraction