Rodrigues Anderson M, Najafzadeh Mohammad J, de Hoog G Sybren, de Camargo Zoilo P
Cell Biology Division, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo São Paulo, Brazil.
Department of Parasitology and Mycology, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences Mashhad, Iran.
Front Microbiol. 2015 Dec 8;6:1385. doi: 10.3389/fmicb.2015.01385. eCollection 2015.
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 × 10(6) 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.
在原本健康的患者中，孢子丝菌感染正成为一种对人类和动物的重要威胁，尤其是在巴西和中国。正确鉴定孢子丝菌病病原体有利于流行病学监测，有助于实施适当的公共卫生政策并指导抗真菌治疗。在孢子丝菌病流行的资源有限地区，特异性和敏感性高的高通量检测方法优于通常会导致密切相关的孢子丝菌物种误判的表型方法。我们试图将滚环扩增（RCA）确立为一种低成本的筛查工具，用于对人类致病性孢子丝菌进行物种特异性鉴定。我们开发了六种针对钙调蛋白编码基因多态性的物种特异性锁式探针。BLAST搜索揭示了种内保守的候选探针；未发现与孢子丝菌属成员以外的人类、小鼠、植物或微生物序列有显著同源性。通过探针 - 模板与25株巴西孢子丝菌、58株申克孢子丝菌、5株球形孢子丝菌、1株卢里孢子丝菌、4株墨西哥孢子丝菌和3株苍白孢子丝菌样本的特异性结合，证明了我们基于RCA的检测方法的准确性。在体外，密切相关物种之间未出现明显的交叉反应，锁式探针产生了100%的特异性和低至3×10⁶个靶序列拷贝的敏感性。基于RCA的物种形成与通过对钙调蛋白编码基因和rDNA操纵子进行系统发育分析的鉴定结果相匹配（kappa 1.0；95%置信区间1.0 - 1.0），支持其作为DNA测序的可靠替代方法。该方法是快速鉴定和特异性检测医学相关孢子丝菌的有力工具，并且由于其稳健性，具有用于生态学研究的潜力。
