new multiplex PCR for species-specific diagnosis of human candidiasis

Liliana Torcoroma García, Liany Johanna Luna, Tania Katherine Velasco, Beatriz Elena Guerra

Candidiases is a group of opportunistic infections caused by yeasts belonging to the genus Candida. Candida albicans is the most prevalent species in both superficial and deep infections, however, the clinical importance of non-albicans Candida has increased during the last decade, driving an urgent need for diagnostic tests that allow for species-level resolution and selection of the optimum therapeutic approach. To design and to optimize a new multiplex PCR assay for the simultaneous identification of the five most relevant species of Candida involved in human candidiasis etiology. For primers design, the physical and thermodynamic restrictions that affect multiplex PCR performance were analyzed using Gene Runner and Mult-PSOS. As templates, the internal transcribed region 2 (ITR2) was selected for C. albicans (AJ249486.1), and topoisomerase II (TOPII) for C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1), and C. guillermondii (AB049145.1). We used ATCC strains of all these five species and clinical isolates as templates. We designed ten oligonucleotides for the simultaneous amplification of the Candida species. The electrophoresis band profile was: C. albicans (206 bp), C. guillermondii (244 bp), C. tropicalis (474 bp), C. parasilopsis (558 bp), and C. krusei (419 bp). The new multiplex PCR assay designed in this study allowed a simultaneous and efficient amplification of the amplicons corresponding to the five species of Candida under study, with an adequate resolution in standard agarose gel.