SINGLE-TUBE, NESTED PCR FOR THE DIAGNOSIS OF POLYMYXA-BETAE INFECTIONIN SUGAR-BEET ROOTS AND COLORIMETRIC ANALYSIS OF AMPLIFIED PRODUCTS

Nested primers for the specific amplification of DNA sequences from th e obligate parasitic root-infecting fungus Polymyxa betae in a single- tube reaction are described. The choice of primers, DNA purity, and re lative concentration of outer to inner primers were critical to the su ccess of single-tube reactions. The polymerase chain reaction (PCR) te st discriminated against background DNA from the host plant and contam inating microorganisms and detected P. betae in as little as 1 pg of t otal genomic DNA from infected roots. For rapid analysis of amplified products, primers were modified to generate products that could be det ected in a colorimetric assay with the commercially available Captagen e-GCN4 kit. It was essential to design a PCR protocol that reduced pri mer dimerization to levels that did not lead to high background absorb ance readings. Results from the Captagene-GCN4 test were compared to t hose obtained by agarose gel analysis of PCR products.