Biological Systems: Theory and Innovation

Bioinformatic design and pilot validation of a PCR assay for species-level identification of Candida spp. in biological samples

Dmytro Prokhorenko*

dmtr.prokhorenko@outlook.com

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Abstract

Species-level identification of representatives of the genus Candida in complex biological samples of natural, agrobiological and technological origin is necessary for the accurate analysis of mixed microbial communities, the control of microbial contamination and the assessment of potential sources of fungal pathogen dissemination. This study aimed to provide bioinformatic justification, design and preliminary laboratory verification of a polymerase chain reaction system for the species‑level differentiation of Candida glabrata, Candida parapsilosis and Candida auris. The study included analysis of the ribosomal cluster, selection of reference sequences, in silico design of primers and hydrolysis probes, assessment of their specificity, optimisation of the reaction mixture and testing on control samples of DNA and archived isolates of biological origin. The internal transcribed spacer region of the ribosomal cluster was selected as the main target, as it combines conserved flanking fragments with variable internal sequences. Primer-probe sets were developed for Candida glabrata, Candida parapsilosis and Candida auris, with amplicon sizes of 82, 91 and 118 base pairs, respectively. The amplification reaction was performed in a final volume of 25 µl. The final concentration of each primer was 0.28 µM; the concentration of the hydrolysis probe was 0.2 µM, MgCl₂ 3 mM, dNTPs 0.3 mM and betaine 1.5 M. Positive amplification was obtained for 40 positive control samples, including 29 Candida spp. samples, 7 Candida glabrata samples and 4 Candida parapsilosis samples. No cross-reactivity was detected in the negative control panel, which included 43 samples of non-target microorganisms. The proposed system confirmed its suitability for primary qualitative analytical species-level identification of selected representatives of the genus Candida under controlled laboratory conditions. The developed approach may serve as a basis for further sanitary and biological screening of complex matrices of soil, aquatic and plant microbiota, for biosafety control in the agro-industrial sector, and for subsequent adaptation to the early molecular detection of fungal pathogens relevant to invasive infections

Keywords

ribosomal cluster; yeast-like fungi; probes; screening; microbial contamination; microorganisms

Suggested citation
Prokhorenko, D. (2026). Bioinformatic design and pilot validation of a PCR assay for species-level identification of Candida spp. in biological samples. Biological Systems: Theory and Innovation, 17(3), 38-52. https://doi.org/10.31548/biologiya/3.2026.38
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