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

Abstract

Summary 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.

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for Xiphinema diversicaudatum, X. index and X. vuittenezi

“…However, they are impractical for use in a phytosanitary/quarantine environment as the methodologies are time-consuming relative to single-step PCR tests, thus consequently more expensive in terms of both human and financial resources. In the future, quantification PCR assays will be utilised but there are currently few such protocols for nematodes (Bates et al, 2002;Holeva et al, 2002).…”

Chorismate mutase: an alternatively spliced parasitism gene and a diagnostic marker for three important Globodera nematode species

“…Various ITS-based molecular methods including recently developed qPCR assays have been proven to be valuable for identifying Globodera species (Bates et al 2002;Madani et al 2008). Although these qPCR assays offer rapid, specific, and sensitive detection, they utilize limited nucleotide polymorphisms for species differentiation.…”

“…Molecular diagnostic methods utilizing ribosomal DNA (rDNA) sequences have been developed for identifying these three important Globodera species (Bates et al 2002;Bulman and Marshall 1997;Madani et al 2008;Skantar et al 2007;Thiéry and Mugniéry 1996). The species-specific PCR primers developed by Bulman and Marshall (1997) have been found to be useful in differentiating PCN species and species-specific PCR primers for TCN identification were also recently made available (Skantar et al 2007).…”

“…The species-specific PCR primers developed by Bulman and Marshall (1997) have been found to be useful in differentiating PCN species and species-specific PCR primers for TCN identification were also recently made available (Skantar et al 2007). More recently, quantitative real-time PCR (qPCR) assays that include melting curve analysis of PCR products or TaqMan technology utilizing locked nucleic acids (LNA)-modified primers and probes, have been developed for identifying Globodera species (Bates et al 2002;Madani et al 2008). qPCR-based diagnostics offer rapid, specific, and sensitive detection compared to conventional PCR assays that require post-PCR gel analysis.…”

Molecular Diagnostic Tools for Nematodes

“…A multiplex PCR was presented by Bulman and Marshal, (Figure 2), when species-speciic primers were used and combined with mixed populations of PCNs [17]. A few years later, the PCR method was named conventional PCR (CoPCR) due the appearance of quantitative real-time PCR (qPCR) [21].…”