Direct DNA extraction for PCR-mediated assays of soil organisms.
By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
T
T
Volossiouk
E J
EJ
Robb
R N
RN
Nazar
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics; D003904:Mitosporic Fungi / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal