Nested primers for the specific amplification of DNA sequences from the obligate parasitic root-infecting fungus Polymyxa betae in a single-tube reaction are described. The choice of primers, DNA purity, and relative concentration of outer to inner primers were critical to the success of single-tube reactions. The polymerase chain reaction (PCR) test discriminated against background DNA from the host plant and contaminating microorganisms and detected P. betae in as little as 1 pg of total genomic DNA from infected roots. For rapid analysis of amplified products, primers were modified to generate products that could be detected in a colorimetric assay with the commercially available Captagene-GCN4 kit. It was essential to design a PCR protocol that reduced primer dimerization to levels that did not lead to high background absorbance readings. Results from the Captagene-GCN4 test were compared to those obtained by agarose gel analysis of PCR products.
|Number of pages||5|
|Publication status||Published - May 1996|
- DNA-BINDING PROTEINS
- beet necrotic yellow vein virus
- plant breeding
- obligate parasite