四对孢子丝菌引物的特异性和敏感性评价

目的 筛选对孢子丝菌特异且敏感的引物。方法 以50株不同地区来源的孢子丝菌临床分离株及标准株的基因组DNA作为研究样本，以毛霉、烟曲霉、念珠菌临床分离菌株基因组DNA作为对照，通过特异引物PCR扩增的方法筛选国内外文献中报道的4对孢子丝菌引物。所有受试孢子丝菌菌株均出现同一扩增产物，而对照菌株未出现者记为特异性引物，随后将基因组DNA模板倍比稀释后依次行PCR扩增，记录各引物出现阳性扩增结果时的最小模板浓度，检测敏感性。结果 在相同PCR条件下，引物S2-R2、SSHF31-SSHR97及ITS3-SSP具有较好特异性，而引物SS3-SS4对孢子丝菌及念珠菌菌株均可扩增出同样大小的PCR产物。引物S2-R2的敏感性最好，基因组DNA模板浓度为5 pg/μL时即可被检出。结论 针对几丁质合成酶1基因的引物S2-R2为孢子丝菌特异且敏感的引物。

Sensitivity and specificity evaluation of four primers for Sporothrix schenckii

Objective To evaluate the sensitivity and specificity of primers for Sporothrix schenckii. Methods Genomic DNA was extracted from 50 strains of S.schenckii, including 49 clinical isolates and 1 referrence strain, as well as from 1 strain of Mucor, Aspergillus fumigatus and Candida albicans, respectively, as controls. Four primers were designed according to previous studies, and screened by PCR. Specific primers were defined as those capable of yielding the same DNA fragment from all strains of S.schenckii, but not from any control fungal strains. Then, the genomic DNA was proportionally diluted and subjected to PCR amplification. The minimal concentration of template DNA required for efficient amplification was determined for the sensitivity assay of primers. Results Under the same PCR conditions, three primers, including S2-R2, SSHF31-SSHR97 and ITS3-SSP, had a relatively high specificity. PCR with the primer SS3-SS4 amplified the same DNA fragment from S.schenckii and C.albicans strains. The primer S2-R2 had the highest sensitivity with a detection limit of 5 pg/μL. Conclusion So far, the primer S2-R2 targeting the chitin synthase 1 gene is the most specific and sensitive primer for S.schenckii .