Rapid identification of emerging human-pathogenic Sporothrix species with rolling circle amplification

Rodrigues, A. M. and Najafzadeh, M. J. and De Hoog, G. S. and De Camargo, Z. P. (2015) Rapid identification of emerging human-pathogenic Sporothrix species with rolling circle amplification. Frontiers in Microbiology, 6 (DEC).

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Abstract

Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and guiding antifungal therapy. In areas of limited resources where sporotrichosis is endemic, high-throughput detection methods that are specific and sensitive are preferred over phenotypic methods that usually result in misidentification of closely related Sporothrix species. We sought to establish rolling circle amplification (RCA) as a low-cost screening tool for species-specific identification of human-pathogenic Sporothrix. We developed six species-specific padlock probes targeting polymorphisms in the gene encoding calmodulin. BLAST-searches revealed candidate probes that were conserved intraspecifically; no significant homology with sequences from humans, mice, plants or microorganisms outside members of Sporothrix were found. The accuracy of our RCA-based assay was demonstrated through the specificity of probe-template binding to 25 S. brasiliensis, 58 S. schenckii, 5 S. globosa, 1 S. luriei, 4 S. mexicana, and 3 S. pallida samples. No cross reactivity between closely related species was evident in vitro, and padlock probes yielded 100 specificity and sensitivity down to 3 × 106 copies of the target sequence. RCA-based speciation matched identifications via phylogenetic analysis of the gene encoding calmodulin and the rDNA operon (kappa 1.0; 95 confidence interval 1.0-1.0), supporting its use as a reliable alternative to DNA sequencing. This method is a powerful tool for rapid identification and specific detection of medically relevant Sporothrix, and due to its robustness has potential for ecological studies. © 2015 Rodrigues, Najafzadeh, de Hoog and de Camargo.

Item Type: Article
Additional Information: Cited By :16 Export Date: 16 February 2020 Correspondence Address: Rodrigues, A.M.; Cell Biology Division, Department of Microbiology, Immunology and Parasitology, Federal University of São PauloBrazil; email: amrodrigues.amr@gmail.com
Uncontrolled Keywords: Calmodulin Diagnostics Epidemiology Identification PCR RCA Sporothrix Sporotrichosis fungal DNA Article diagnostic accuracy DNA sequence fungal strain fungus identification gene amplification limit of detection molecular phylogeny nonhuman nucleic acid probe phylogenetic tree rapid test rolling circle amplification sensitivity and specificity sequence analysis
Subjects: QX Parasitology
Divisions: Mashhad University of Medical Sciences
Depositing User: mr lib5 lib5
Date Deposited: 22 Apr 2020 07:22
Last Modified: 22 Apr 2020 07:22
URI: http://eprints.mums.ac.ir/id/eprint/16574

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