, 2008; Varillas et al., 2010), bacteria (Li et al., 2010; Robertson et al., 2010; Šimenc & Potočnik, 2011), nematodes (Holterman et al., 2012), and fungi (Ricchi et al., 2011). The recent development of better performing saturating DNA dyes and the technical progress that enabled the increased resolution and precision of the instruments have permitted the use of HRM for genotyping (Ganopoulos et al., 2011a, b). Although HRM is a very sensitive technique, the risk of contamination is significantly KPT-330 order reduced
compared to multi-step procedures, such as RFLP or nested PCR, because the entire process is completed in a single closed tube. The objective of this study was to develop and validate the HRM method for F. oxysporum formae speciales complex identification based on differences in melting curve characteristics via the ITS of the ribosomal DNA. The results presented in this study show that HRM curve analysis of Fusarium ITS sequences is a simple, quick, and reproducible method that
allows the identification of seven F. oxysporum formae speciales. Seven isolates of F. oxysporum formae speciales (F. oxysporum f. sp. phaseoli, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. radicis-lycopersici, F. oxysporum f. sp. melonis, F. oxysporum, F. oxysporum f. sp. dianthi, and F. oxysporum f. sp. vasinfectum) were analyzed with HRM analysis (Table 1). In addition, two Ipilimumab research buy fungal isolates of Verticillium dahliae and Thielaviopsis basicola that cause cotton vascular wilt disease and black root rot respectively were included in this study as out group (data not shown). Genomic fungal DNA was extracted according to Zambounis et al. (2007). DNA concentrations were determined spectrophotometrically and/or by quantitation on agarose gels stained with ethidium bromide in comparison with molecular marker λ-DNA-HindIII (Gibco-BRL, Gaithersburg, MD). PCR amplification, DNA melting, and end point fluorescence FAD level acquiring PCR amplifications were performed in a total volume
of 15 μL on a Rotor-Gene 6000 real-time 5P HRM PCR Thermocycler (Corbett Research, Sydney, Australia) according to Ganopoulos et al. (2011a, b). Universal primers, ITS1 (5′-tccgtaggtgaacctgcgg-3′) and ITS4 (5′-tcctccgcttattgatatgc-3′), specific for the internal transcribed spacer of the rDNA were used to generate amplicons (c. 570 bp; White et al., 1990). More specifically, the reaction mixture contained 20 ng genomic DNA, 1× PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 300 nM forward and reverse primers, 1.5 mM Syto® 9 green fluorescent nucleic acid stain, and 1 U Kapa Taq DNA polymerase (Kapa Biosystems). A rapid PCR protocol was conducted in a 36-well carousel using an initial denaturing step of 95 °C for 3 min followed by 35 cycles of 95 °C for 20 s, 55 °C for 45 s and 72 °C for 50 s, then a final extension step of 72 °C for 2 min. The fluorescent data were acquired at the end of each extension step during PCR cycles.