HRM was performed as described previously by Ganopoulos et al (2

HRM was performed as described previously by Ganopoulos et al. (2011b). Each formae speciales was set as a ‘genotype’ (reference), and the average HRM genotype confidence percentages (GCPs; value attributed to each formae speciales being compared to the genotype, with a value of 100 indicating an exact match) for the replicates (disregarding the most outlying replicate) were tabulated (Hewson et al., 2009). PCR products were analyzed on a 1% agarose gel to ensure the amplification of the correct size products. All the experiments were repeated three times with three independent samples. Figure 1a presents the data analysed by means of conventional derivative plots in the ‘genotyping’ mode. It shows that NVP-BKM120 concentration each genotype

was represented by two peaks, except for F. oxysporum f. sp. dianthi which was represented by three peaks. The first peak ranged from 85.15 to 85.45 °C, the second peak from 88.37 to 89.32 °C, and the third peak was 90.70 °C (Table 2). The different formae speciales tested generated distinctive HRM profiles and normalized HRM profiles, allowing the discrimination selleck chemicals and differentiation

of each species. The potential resolving power of this approach is much greater than conventional melting curve analysis because, in HRM, melting curves from different amplicons can be differentiated on the basis of shape even when they define the same T m values as a result of the composite melting curves of heterozygotes (Ganopoulos et al., 2011b). In this study, we have used the shape of the melting curves, which is more informative, to assess differences in the formae speciales under investigation (Fig. 1b). Analysis of Isotretinoin the normalized HRM

curves produced with the ITS marker revealed that most of the formae speciales could easily be distinguished, for instance for ‘F. oxysporum f. sp. lycopersici’ and ‘F. oxysporum f. sp. melonis’, the curve profiles of some formae specials were similar and could therefore not be visually differentiated. Furthermore, closer examination of the F. oxysporum f. sp. lycopersici’ differentiation curve, with the mean F. oxysporum f. sp. vasinfectum curve as the baseline, revealed part of the curve sitting outside the 90% CI curve, suggesting that all the examined formae speciales via the HRM curves are indeed different (Fig. 1b). Assigning the ‘F. oxysporum f. sp. lycopersici’ as a genotype, we were able to estimate the confidence value of similarity between F. oxysporum f. sp. lycopersici and the other formae speciales used in the study and to show that ITS was a sufficient region to distinguish the tested formae speciales (Fig. 1c). The average GCPs resulting from HRM analysis of the ITS region of seven F. oxysporum formae speciales are shown in Table 3. GCPs were calculated, and a cutoff value of 90% was used to assign a genotype for each region. The highest GCP (82.63) was found between the F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp.

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