Use of Rolling Circle Amplification to Rapidly Identify Species of Cladophialophora Potentially Causing Human Infection

Abstract

The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis

“…The RCA padlock probe (FPgP) in the present study demonstrated greater sensitivity than previously reported probes developed for Fonsecaea species [13,15]. Given the specificity of the RCA padlock probes, RCA was judged to be suitable for Fonsecaea detection and species differentiation without sequencing in a wide range of biological samples.…”

“…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].…”

“…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].…”

Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis

“…13,14 RCA, a technique based on the rolling replication of short, single-stranded DNA circles by DNA polymerases under isothermal conditions, has been shown to be rapid, cost-effective and specific for molecular identification of human-and plant-pathogenic fungi, [15][16][17][18][19] including black yeasts and their relatives. [20][21][22][23] Kong et al 16 developed a rapid (2 h) and specific RCA-based single-nucleotide polymorphism detection assay for identification of clinically important Trichophyton species.…”

“…RCA, a technique based on the rolling replication of short, single‐stranded DNA circles by DNA polymerases under isothermal conditions, has been shown to be rapid, cost‐effective and specific for molecular identification of human‐ and plant‐pathogenic fungi, including black yeasts and their relatives . Kong et al .…”