Single-tube, nested PCR for the diagnosis of Polymyxa betae infection in sugar beet roots and colorimetric analysis of amplified products

Abstract

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.