High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

The fungus Fusarium oxysporum is a highly complex species composed by many strains put together into groups called formae speciales. As it is difficult and laborious to discriminate Fusarium formae specials via biochemical or phenotypic methods, it is very important to develop novel, rapid, and simple to perform identification methods. Herein, real-time PCR assay [using universal internal transcribed spacer (ITS) primers] coupled with high-resolution melting (HRM) analysis was developed for identifying and distinguishing F.similar to oxysporum formae speciales complex. The melting curve analysis of these amplicons specifically classified all isolates into seven F.similar to oxysporum formae speciales and generated seven HRM curve profiles. The smallest DNA sequence difference recognized in this study was one nucleotide. The results presented show that HRM curve analysis of Fusarium ITS sequences is a simple, quick, and reproducible method that allows both the identification of seven F.similar to oxysporum formae speciales and at the same time their screening for variants. Our genotyping assay uses the combined information of simultaneously acquired HRM data from an unlabeled probe and the full-length amplicon. Finally, the completion of both reaction and analysis in a closed tube saves time by eliminating the separate steps and reduces the risk of contamination.