Department of Dermatology, No1 Hospital of China Medical University, Shenyang, China.
Med Mycol. 2010 Feb;48(1):161-5. doi: 10.3109/13693780903117481.
We employed a nested PCR assay to detect Sporothrix schenckii DNA of 38 strains (including all the 24 mtDNA types) collected from different areas of the world, in tissue of eight mice infected with ATCC10268 strain of the fungus, and the skin biopsies of nine patients with sporotrichosis. In addition, the same procedures were used with two strains of Ceratocystis minor and isolates of 10 species of other pathogenic fungi. The outer primers SS(1) and SS(2) and inner primers SS(3) and SS(4) of the 18S rRNA gene of S. schenckii were employed. A 152 bp fragment was detected in all 38 strains of S. schenckii, eight animal specimens and nine human skin biopsies, but not samples of C. minor and the other fungal species. The detection limit of 50 fg of S. schenckii DNA extract was determined with ethidium bromide staining. In summary, we demonstrated that nested PCR assay could identify S. schenckii of all the mtDNA types and in isolates recovered from different areas of the world. The nested PCR assay seems to be highly sensitive and specific and provides a rapid method for diagnosis of sporotrichosis under conditions of contamination avoidance.
我们采用巢式 PCR 检测方法，对来自世界各地不同地区的 38 株（包括全部 24 种线粒体 DNA 型）申克孢子丝菌进行检测，所用菌株分别为感染 ATCC10268 株真菌的 8 只小鼠的组织和 9 例孢子丝菌病患者的皮肤活检样本。此外，我们还用两种小丛壳菌和 10 种其他致病性真菌的分离株，进行了同样的检测。我们采用了申克孢子丝菌 18S rRNA 基因的外引物 SS(1)和 SS(2)以及内引物 SS(3)和 SS(4)。在所有 38 株申克孢子丝菌、8 个动物标本和 9 例人皮肤活检样本中均检测到 152bp 片段，但小丛壳菌和其他真菌分离株未检测到该片段。采用溴化乙锭染色法，确定了检测 50fg 申克孢子丝菌 DNA 提取液的最低检出限。总之，我们的研究表明，巢式 PCR 检测方法可鉴定出所有线粒体 DNA 型的申克孢子丝菌，以及来自世界各地不同地区的分离株。该巢式 PCR 检测方法似乎具有高度的敏感性和特异性，可在避免污染的情况下，为孢子丝菌病的诊断提供一种快速方法。
