Sandhu G S, Kline B C, Stockman L, Roberts G D
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Clin Microbiol. 1995 Nov;33(11):2913-9. doi: 10.1128/jcm.33.11.2913-2919.1995.
We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.
我们已开发出21种特异性核酸探针，其靶向黄曲霉、烟曲霉、灰绿曲霉、黑曲霉、土曲霉、皮炎芽生菌、白色念珠菌、光滑念珠菌（都柏林念珠菌）、季也蒙念珠菌、克柔念珠菌、葡萄牙念珠菌、近平滑念珠菌、热带念珠菌、粗球孢子菌、新型隐球菌格特变种、新型隐球菌新生变种、新生隐球菌芽孢变种、新生隐球菌新生变种、荚膜组织胞浆菌、波伊德假阿利什霉和申克孢子丝菌的大亚基rRNA基因。对约100种真菌（代表约50种病原体和常见腐生菌）的28S rRNA基因的一部分进行了测序，以开发通用PCR引物和种特异性寡核苷酸探针。检测和鉴定过程中的每一步都被标准化为一组通用条件，无需修改即可适用于所有感兴趣的真菌和所有类型的临床标本。这些步骤包括在碱性胍-苯酚- Tris试剂中煮沸标本进行DNA提取、用通用引物扩增28S rRNA基因的可变区以及通过在所有真菌相同条件下进行的探针杂交或DNA测序来鉴定扩增子。通过检测一组真菌分离株和各种临床标本获得的结果表明具有高度特异性。
