A quest to find good primers for gene expression analysis of Candida albicans from clinical samples

Autor(es)

Alonso, Gabriela C. ^[1] ; Pavarina, Ana C. ^[1] ; Sousa, Tabata V. ^[1] ; Klein, Marlise I. ^[1]

Afiliação do(s) autor(es)

^[1] Sao Paulo State Univ, UNESP, Dept Dent Mat & Prosthodont, Sch Dent, Rua Humaita 1680, BR-14801903 Araraquara, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Microbiological Methods; v. 147, p. 1-13, APR 2018.
• Citações Web of Science: 3

Resumo

Biofilm production contributes to several human diseases, including oral candidiasis. Among the Candida species, Candida albicans is the most prevalent. The expression of virulence genes is implicated in the pathogenic potential of Candida biofilms. However, the evaluation of microbial gene expression from in vivo biofilm samples is not trivial, specifically, assessment via quantitative PCR (qPCR) can be a challenge because of several species present in clinical samples. Hence, the necessity of primers specificity. The aim of this study was to evaluate through in silico and in vitro analyses the specificity of published primers and newly designed primers for C. albicans virulence genes: ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, SAP4, SOD1, SODS and ACT1 (normalizing gene). In silico analysis was performed through a PubMed search of articles with primer sequences that evaluated gene expression of C. albicans. Then, the sequence similarity of twenty-eight primers was checked through BLASTn and ClustalW2. The analysis of secondary structures was performed using mfold. When the primers did not present satisfactory characteristics (absence of secondary structures, not discrepant Tm of forward and reverse sequences and specificity) following in vitro analysis (i.e., end point PCR), new primers were designed using Beacon Designee{''} and sequences obtained from the ``Candida Genome Database{''}. The selected primers were tested in vitro by end point PCR using a panel of genomic DNA from five different Candida species (C. albicans, Candida glabrata, Candida dubliniensis, Candida krusei, and Candida tropicalis). The resulting PCR products were visualized on agarose gel. qPCR reactions were performed to determine primers' optimal concentration and PCR efficiency. End point PCR demonstrated that published primers for the SAP1 and HWP1 were specific for C. albicans and the one for SOD1 reacted with C. albicans and C. dubliniensis. The sequence of primers designed for ACT1, ALS1 and HWP1 genes were specific for C. albicans, while the ones for CAP1, CAT1, EFG1, LIP3, and PLB1 were detected in C. albicans and C. dubliniensis. After optimization, all primers presented a single peak on melt curves, correlation coefficient of congruent to 1 and qPCR reaction efficiency of 90-110%, with slope of congruent to-3.3. Therefore, these primers should be suitable for future gene expression analyses from clinical samples. (AU)

• Processo FAPESP: 13/07276-1 - CEPOF - Centro de Pesquisa em Óptica e Fotônica
• Beneficiário: Vanderlei Salvador Bagnato
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 15/13409-0 - Expressão de genes de virulência de isolados clínicos de Candida albicans submetidos à Terapia Fotodinâmica Antimicrobiana
• Beneficiário: Gabriela Caroline Alonso
• Modalidade de apoio: Bolsas no Brasil - Mestrado