Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes

Abstract

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Keywords: Black yeasts; Identification; Rolling circle amplification; Waterborne Exophiala.

Fig. 1

Proof of species specificity of RCA padlock probes and intraspecific variation of RCA response. Amplification and subsequent fluorescent banding were seen only with appropriate template–probe mixtures (Empty lanes denote the absence of signals with unmatched template–probe mixtures.) The species-specific probes are labeled as listed in Table 1 (Equi, E. equina; Esal, E. Salmonis; Eopp, E. opportunistica; Epis, E. pisciphila; Eagu, E. aquamarina; Vbot, V. botryosa; Eang, E. angulospora; Ecas, E. castellanii) lanes: M is 200-bp DNA MW marker (Eurogentec, the Netherlands); 1 to 8, RCA reaction with DNA of E. equina (CBS 109879) (lane 1), E. Salmonis (CBS 110371) (lane2), E. opportunistica (CBS 631.69) (lane 3), E. pisciphila (CBS 119913) (lane 4), E. aquamarina (CBS 119915) (lane 5), V. botryosa (CBS 121506) (lane 6), E. angulospora (CBS 119911) (lane 7) and E. castellanii (CBS 110025) (lane 8)