Alonso Gabriela C, Pavarina Ana C, Sousa Tábata V, Klein Marlise I
Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil.
Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil.
J Microbiol Methods. 2018 Apr;147:1-13. doi: 10.1016/j.mimet.2018.02.010. Epub 2018 Feb 15.
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, SOD5 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 Designer™ 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 ≅1 and qPCR reaction efficiency of 90-110%, with slope of ≅-3.3. Therefore, these primers should be suitable for future gene expression analyses from clinical samples.
生物膜的形成与多种人类疾病有关，包括口腔念珠菌病。在念珠菌属中，白色念珠菌最为常见。毒力基因的表达与念珠菌生物膜的致病潜力有关。然而，评估体内生物膜样本中的微生物基因表达并非易事，具体而言，由于临床样本中存在多种菌种，通过定量聚合酶链反应（qPCR）进行评估可能具有挑战性。因此，引物特异性很有必要。本研究的目的是通过计算机模拟和体外分析，评估已发表的引物以及新设计的针对白色念珠菌毒力基因（ALS1、CAP1、CAT1、EFG1、HWP1、LIP3、PLB1、SAP1、SAP4、SOD1、SOD5和ACT1（标准化基因））的引物的特异性。通过在PubMed中搜索具有评估白色念珠菌基因表达引物序列的文章进行计算机模拟分析。然后，通过BLASTn和ClustalW2检查28种引物的序列相似性。使用mfold进行二级结构分析。当引物在体外分析（即终点PCR）后未呈现令人满意的特性（无二级结构、正向和反向序列的熔解温度（Tm）无差异以及特异性）时，使用Beacon Designer™设计新引物，并从“念珠菌基因组数据库”获取序列。使用来自五种不同念珠菌（白色念珠菌、光滑念珠菌、都柏林念珠菌、克柔念珠菌和热带念珠菌）的基因组DNA面板，通过终点PCR在体外测试所选引物。将所得的PCR产物在琼脂糖凝胶上进行可视化分析。进行qPCR反应以确定引物的最佳浓度和PCR效率。终点PCR表明，已发表的针对SAP1和HWP1的引物对白色念珠菌具有特异性，针对SOD1的引物与白色念珠菌和都柏林念珠菌发生反应。为ACT1、ALS1和HWP1基因设计的引物序列对白色念珠菌具有特异性，而针对CAP1、CAT1、EFG1、LIP3和PLB1的引物在白色念珠菌和都柏林念珠菌中均被检测到。优化后，所有引物在熔解曲线上均呈现单峰，相关系数约为1，qPCR反应效率为90 - 110%，斜率约为 - 3. .3。因此，这些引物应适用于未来临床样本的基因表达分析。
