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.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis

“…Currently, non-culture methods are being used to improve the sensitivity and specificity of mycological diagnosis [29]. Several studies have been published on the identification of specific CBM agents [12,14,30]. Although the ITS region efficiently detects specific causative agents, it is not always recommended because molecular identification may be hampered by sequence variability in the ITS domain caused by difficult-to-sequence homopolymeric regions [25].…”

“…In general, applicability of RCA padlock probe can range from bioprospecting, biotechnological potential [37], and biosensing [38] to diagnostic of viral [39] and bacterial [40] infections. Besides, some studies have already distinguished several CBM agents of the genus F. pedrosoi, F. nubica [10], and C. carrionii [13] and associated CBM species such as Exophiala [11,12] and Cyphellophora [43]. Several studies have reported the application of the RCA padlock probe to detect species-specific fungal diseases with great public impact, e.g., in opportunistic infections caused by species of Candida [16], Aspergillus [16], and Cryptococcus [7,41]; agents associated with systemic infections such as Histoplasma [18], Fusarium [42] and Talaromyces marneffei [15]; and dermatophytoses caused by species of the genus Trichophyton [17].…”

“…Exonucleolysis was used to remove the unligated padlock probes and template PCR products to reduce ligation-independent amplification events. This was performed in a 10 lL reaction mixture containing 10 U each of exonucleases I and III (New England Biolabs; Hitchin, UK) with incubation at 37°C for 30 min, followed by 94°C for 3 min to inactivate the exonuclease reaction [12].…”

“…Likewise, padlock probes have been used in rolling circle amplification (RCA) to supplement the diagnosis of several mycotic diseases [10]. These methods demonstrated good reproducibility and sensitivity with CBM agents such as F. monophora, F. nubica, F. pedrosoi, Cladophialophora, and Exophiala [11][12][13]. Moreover, the method has also been applied for the diagnosis of infections caused by Candida, Penicillium, Aspergillus, Scedosporium, Cryptococcus, and Trichophyton [14][15][16][17].…”

Environmental prospecting of black yeast-like agents of human disease using culture-independent methodology

“…ITS, TEF1, BT2, and ACT1 19 . Alternatively, padlock probes, which are specific oligonucleotides with the ability to identify single nucleotide polymorphisms (SNPs), have been proposed for the recognition of several groups of black agents [20][21][22][23][24][25] . DNA barcoding, based on the ITS region and applying short sequences (25-41 bp) of nucleotides specific for a single taxonomic species 26 , can additionally be used to recognize herpotrichiellaceous species by variable regions in the ribosomal operon.…”