Sporotrichosis: In silico design of new molecular markers for the Sporothrix genus

Sporotrichosis, a cosmopolitan mycosis caused by dimorphic fungi of the Sporothrix complex, affects humans and animals. This study aimed to develop new molecular markers for Sporothrix genome detection in biological samples using PCR.

Methods:

A specific region of DNA sequences from the Sporothrix genus, publicly available in GenBank, was chosen for primer design. After testing the in silico specificity of these primers, in vitro specificity was evaluated using the PCR technique.

Results:

Three specific primers with 100% specificity for the Sporothrix genus were generated.

Conclusions:

PCR using the designed primers can be used to develop molecular diagnostics for sporotrichosis.

Keywords:
Sporothrix sp; Polymerase chain reaction; Sporotrichosis; Molecular diagnosis

Authorship

Blenda Fernandes

Study design

Data collection

Data analysis

Writing

Hospital Santa Casa de Belo Horizonte, Ensino e Pesquisa, Belo Horizonte, MG, Brasil.Hospital Santa Casa de Belo HorizonteBrasilBelo Horizonte, MG, Brasil Hospital Santa Casa de Belo Horizonte, Ensino e Pesquisa, Belo Horizonte, MG, Brasil.

https://orcid.org/0000-0002-0827-1134

Emanoelle Fernandes Rutren La Santrer

Data collection

Writing

https://orcid.org/0000-0002-6415-7758

Sônia Maria de Figueiredo

Data collection

Writing

Universidade Federal de Ouro Preto, Escola de Nutrição, Programa de Pós-Graduação em Saúde e Nutrição, Ouro Preto, MG, Brasil.Universidade Federal de Ouro PretoBrasilOuro Preto, MG, Brasil Universidade Federal de Ouro Preto, Escola de Nutrição, Programa de Pós-Graduação em Saúde e Nutrição, Ouro Preto, MG, Brasil.

https://orcid.org/0000-0001-8149-5110

Fabiana Rocha-Silva

Data collection

Data analysis

https://orcid.org/0000-0003-2698-9649

Cláudia Barbosa Assunção

Study design

Data analysis

https://orcid.org/0000-0001-5530-9310

Amanda Gabrielle Abreu

Data analysis

https://orcid.org/0000-0003-1251-1660

Iara Furtado Santiago

Study design

Writing

Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Microbiologia, Belo Horizonte, MG, Brasil.Universidade Federal de Minas GeraisBrasilBelo Horizonte, MG, Brasil Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Microbiologia, Belo Horizonte, MG, Brasil.

https://orcid.org/0000-0001-5324-4445

Susana Johann

Study design

Writing

https://orcid.org/0000-0001-8068-1720

Rachel Basques Caligiorne

Study design

Data collection

Data analysis

Writing

https://orcid.org/0000-0001-7574-0984

Corresponding author: Dr. Sônia Maria de Figueiredo. e-mail:smfigue@ufop.edu.br

Conflict of interest: The authors have no conflicts of interest to declare and confirm that each has made substantial contributions to the information or materials submitted for publication.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (2)

Thumbnail

FIGURE 1:
A 7% polyacrylamide gel stained with 2% silver nitrate showing the nested-PCR (“In”) amplification using the primer pair SPOR3 (forward) (5′-ATCTCTTGGCTCTGGCATCG-3′) and R4 (reverse) (5′-GGGAGAACATGCGTTCGGTA-3′), with 1 µL of the amplicon of the first PCR (“Out”), diluted to 1/10, as a DNA template. The fragment amplified was approximately 260 bp, as observed via computational tool analysis. Lane 1: molecular weight (MW) standard of 100 base pairs (bp); lane 2: DNA extracted from an international standard sample of Sporothrix schenkii ATCC MF765977 as a positive control; lanes 3-10: DNA extracted from lesion biopsies of patients with clinically and laboratory-confirmed sporotrichosis, at Santa Casa Hospital; lane 11: negative control (CN).

Thumbnail

FIGURE 2:
A 7% polyacrylamide gel stained with 2% silver nitrate showing the nested-PCR (In) amplification using the primer pair SPOR3 (forward) (5′-ATCTCTTGGCTCTGGCATCG-3′) and R4 (reverse) (5′-GGGAGAACATGCGTTCGGTA-3′), with 1 ?#61549;L of the amplicon of the first PCR (“Out”), diluted to 1/10, as the DNA template. The fragment amplified was approximately 260 bp, as observed via computational tool analysis. For the PCR specificity test, strains of different fungal species were analyzed. Lane 1: molecular weight standard, 100 bp ladder; lane 2: DNA extracted from an international standard sample of Sporothrix schenkii ATCC MF765977 as a positive control for the reaction; lanes 3-4: DNA extracted from biopsies of patients presenting with sporotrichosis confirmed by culture, showing bands of approximately 260 bp; lane 5: DNA extracted from international standard samples of Aspergillus flavus URM7326; lane 6: Aspergillus terreus URM 7309; lane 7: Aspergillus fumigatus URM 7315; lane 8: Candida albicans (ATCC 52A); lane 9: Candida albicans (ATCC 18804); lane 10: Exophiala dermatitidis URM 7460; lane 11: Cryptococcus gattii URM7359; lane 12: Fonsecaea pedrosoi URM3161; lane 13: Paracoccidioides brasiliensis (Pb18); lane 14: Paracoccidioides brasiliensis Pb01.

FIGURE 1: A 7% polyacrylamide gel stained with 2% silver nitrate showing the nested-PCR (“In”) amplification using the primer pair SPOR3 (forward) (5′-ATCTCTTGGCTCTGGCATCG-3′) and R4 (reverse) (5′-GGGAGAACATGCGTTCGGTA-3′), with 1 µL of the amplicon of the first PCR (“Out”), diluted to 1/10, as a DNA template. The fragment amplified was approximately 260 bp, as observed via computational tool analysis. Lane 1: molecular weight (MW) standard of 100 base pairs (bp); lane 2: DNA extracted from an international standard sample of Sporothrix schenkii ATCC MF765977 as a positive control; lanes 3-10: DNA extracted from lesion biopsies of patients with clinically and laboratory-confirmed sporotrichosis, at Santa Casa Hospital; lane 11: negative control (CN).

FIGURE 2: A 7% polyacrylamide gel stained with 2% silver nitrate showing the nested-PCR (In) amplification using the primer pair SPOR3 (forward) (5′-ATCTCTTGGCTCTGGCATCG-3′) and R4 (reverse) (5′-GGGAGAACATGCGTTCGGTA-3′), with 1 ?#61549;L of the amplicon of the first PCR (“Out”), diluted to 1/10, as the DNA template. The fragment amplified was approximately 260 bp, as observed via computational tool analysis. For the PCR specificity test, strains of different fungal species were analyzed. Lane 1: molecular weight standard, 100 bp ladder; lane 2: DNA extracted from an international standard sample of Sporothrix schenkii ATCC MF765977 as a positive control for the reaction; lanes 3-4: DNA extracted from biopsies of patients presenting with sporotrichosis confirmed by culture, showing bands of approximately 260 bp; lane 5: DNA extracted from international standard samples of Aspergillus flavus URM7326; lane 6: Aspergillus terreus URM 7309; lane 7: Aspergillus fumigatus URM 7315; lane 8: Candida albicans (ATCC 52A); lane 9: Candida albicans (ATCC 18804); lane 10: Exophiala dermatitidis URM 7460; lane 11: Cryptococcus gattii URM7359; lane 12: Fonsecaea pedrosoi URM3161; lane 13: Paracoccidioides brasiliensis (Pb18); lane 14: Paracoccidioides brasiliensis Pb01.

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Sociedade Brasileira de Medicina Tropical - SBMT Sociedade Brasileira de Medicina Tropical - SBMT, Núcleo de Medicina Tropical UnB, Sala 43C 70904-970, E-mails: rsbmt@uftm.edu.br | artes.rsbmt@gmail.com | sbmt@sbmt.org.br , WhatsApp: SBMT (61) 9.9192-6496, WhatsApp: RSBMT (34) 9.9996-5807 - Brasília - DF - Brazil
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Sporotrichosis: In silico design of new molecular markers for the Sporothrix genus

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[1] Corresponding author: Dr. Sônia Maria de Figueiredo. e-mail:smfigue@ufop.edu.br