应用巢式PCR检测实验小鼠组织中申克孢子丝菌

目的研究实验小鼠申克孢子丝菌感染的分子生物学鉴定方法，为建立检测人申克孢子丝菌感染的快速、敏感、特异方法提供依据。方法建立申克孢子丝菌感染小鼠模型，应用合成的申克孢子丝菌种特异性引物ITS3-SSP进行巢式PCR扩增小鼠皮损组织内核糖体DNA的ITS2靶序列，将检测结果与标准形态学鉴定结果对比，观察种特异性引物PCR扩增结果的准确程度、敏感性和特异性。结果组织学检查可以从11只感染小鼠尾组织中见到染成紫红色的圆形、卵圆形孢子；第一循环PCR检测实验小鼠申克孢子丝菌，只有3只小鼠呈阳性；而巢式PCR检测申克孢子丝菌特异性DNA，11只实验小鼠中9只呈阳性；而3只对照组小鼠无一呈阳性。结论本实验结果说明巢式PCR结合种特异性引物ITS3-SSP可以有效检出实验小鼠皮损中的申克孢子丝菌，具有一定的敏感性和特异性，尤其是对于组织学检查和真菌培养阴性的标本，本方法是一个很有潜力的替代方法。

Detection of Sporothrix schenckii in experimentally infected mice tissues by a nested PCR assay

Objective To study the molecular biology identification methods of Sporothrix schenkii from experimentally infected mice,and to establish specific,sensitive and reliable method.Methods setting up a mouse model of Sporothrix schenckii infection,Sporothrix schenckii strain specific primers ITS3-SSP was used to amplify the internal transcribed spacer region 2 target sequences of mouse skin lesions tissues ribosome DNA by nested PCR.The results of species-specific primer PCR amplification were compared with that of standard morphological identification to observe the accuracy,sensitivity and specificity.Results Histological examination of 11 mice tail tissues showed round,oval fuchsia spores.Three mice presented positive in the first cycle of PCR detection.However,nested-PCR detection of Sporothrix schenckii-specific DNA give out 9 positive from 11 experimental mice and none were positive in the control group.Conclusion The results show that nested PCR combined with species-specific primers ITS3-SSP can effectively detect Sporothrix schenckii in experimental infected mice lesions and present a certain sensitivity and specificity,especially for histological examination and fungal culture negative specimens.This method is a promising alternative for clinical molecular diagnosis.