Determination of relative proportions of Globodera species in mixed populations of potato cyst nematodes using PCR product melting peak analysis

Using a novel approach based on melting peak analysis of PCR products, we have developed a semi-quantitative assay to measure the relative proportions of Globodera pallida and Globodera rostochiensis in a sample of potato cyst nematodes (PCN). The method depends on a competitive multiplex PCR where the products of each species can be separated by their distinct melting temperatures (T-m). The melting curves of the products are measured by continual fluorescence monitoring in the presence of the intercalating dye SYBR Green I whilst gradually increasing the temperature. Varying the proportion of cysts of each PCN species gave rise to melting curves with different peak heights, which reflected the relative amounts of each DNA in the sample. By calculating the ratio of the melting peak heights at the Tm of each product and comparing it with standards run under the same conditions, it was possible to estimate the proportion of each product in the mixture. Sensitivity was such that 2% of G. pallida cysts in a mixture could be detected. All results predicted by the analysis were confirmed by agarose gel electrophoresis of the PCR products. The method is rapid, reproducible and following DNA extraction, up to 25 samples can be analysed in an hour.