McRAPD - a new approach to rapid and accurate identification of pathogenic yeast species.
Vladislav Raclavsky (1), Jitka Trtkova (1), Lucia Smilkova (1), Anna Konieczna (1), Radek Plachy (2), Petr Hamal (3)
(1) Dept. of Biology, Palacky University, Hnevotinska 3, Olomouc, 775 15, Czech Republic; (2) Haemato-Oncology Ward, University Hospital, I.P.Pavlova 6, 775 20 Olomouc, Czech Republic; (3) Dept. of Microbiology, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
Immunocompromised patients are increasingly infected with yeast species, which can vary greatly in their virulence as well as their susceptibilities to antifungal agents. Therefore, rapid and accurate identification of yeast isolates to species level is essential for effective treatment. Conventional methods are complex and time-consuming in some species, whereas the rapid and accurate genotyping tools are unable to cover the whole spectrum of yeast species in a cost-effective way. We have recently developed a new approach to identification of five of the most medically important Candida species based on melting curve analysis of RAPD products (McRAPD). Here we describe an optimized McRAPD protocol and prove its usefulness in a broader spectrum of pathogenic yeast species. The primer ACGGGCCAGT described previously as suitable for yeast species identification was used and the reaction conditions were optimized to increase the specificity of McRAPD-results. Enzymes and reagents from different suppliers were also tested to asses their suitability for McRAPD. Yeast DNA isolated by a simplified technique described previously was demonstrated to be of sufficient quality for the use in McRAPD. As a result, an optimized McRAPD protocol is presented which allows accurate identification of pathogenic yeast species within 2,5 hours after minute colonies of a pure isolate are available on plate. The Ministry of Health, Czech Republic, supported this work (NR8365-4/2005).