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Background  Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs.  Candida  spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of  Candida  may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with T m  calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant  Candida  species.    Methods  Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified  Candida  EDTA-whole blood (WB) reference panels seeded with  Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis  cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated.    Results  The limit of reliable detection proved to be 0.2–2 genomic equivalent and the method was reliable on broad concentration ranges (10 6 –10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable.    Conclusion  Our assay demonstrates recent advances on T m  calling and HRM analysis for the molecular identification of relevant  Candida  species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12–24 h).    Electronic supplementary material  The online version of this article (10.1186/s12879-018-3283-6) contains supplementary material, which is available to authorized users.

Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species