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

An improved plant and soil DNA extraction method for detection and quantifi cation of Verticillum species using polymerase chain reaction (FCR) based t echniques is presented. This method involves the use of extraction buffer c ontaining proteinase K and further DNA purification with addition of ammoni um acetate. In the case of soils, the protocol combines the benefits of usi ng a commonly reported nucleic acid carrier with the simplicity of the prot einase K - ammonium acetate method. As organic solvent extractions are not needed and the DNA extractions can be performed in small volumes, this meth od becomes a very attractive alternative when a large number of plant tissu e or soil samples have to be processed for PCR detection and (or) quantific ation. In addition, comparative studies with the traditional SDS buffer - p henol 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, r epresents an improved alternative for detection and quantification of Verti cillium spp., in both plant tissue and soil samples.