de Llanos Frutos Rosa, Fernández-Espinar M Teresa, Querol Amparo
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de los Alimentos, CSIC P.O. Box 73, 46100 Burjassot, Valencia, Spain.
Antonie Van Leeuwenhoek. 2004 Apr;85(3):175-85. doi: 10.1023/B:ANTO.0000020154.56649.0f.
The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation. The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae / C. beechii and C. zeylanoides / C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.
核糖体区域跨越内部转录间隔区（ITS1和ITS2）以及5.8S rRNA基因的PCR扩增和后续限制性分析，被用于鉴定念珠菌属的酵母。该方法此前已用于该属一些物种的鉴定，但在本研究中，此应用已用于鉴定和表征更多念珠菌属物种，并特别调查了具有临床和生物技术意义的物种。在念珠菌属物种中，观察到的高变异性，无论是在扩增区域的长度（390至900 bp之间）还是其限制性图谱中，都允许明确鉴定到物种水平，但包括膜状念珠菌、球形念珠菌、大西洋念珠菌、大气念珠菌和嗜油念珠菌在内的一组物种除外，它们需要对26S rRNA基因的D1/D2结构域或5.8S-ITS区域进行测序以进行准确区分。5.8S-ITS限制性分析在区分阿氏念珠菌/丁酸念珠菌、果实念珠菌/香蕉念珠菌、圣玛丽亚念珠菌变种圣玛丽亚念珠菌/比氏念珠菌以及锡兰念珠菌/克氏念珠菌这几对物种时也失败了。在这种情况下，它们的26S D1/D2结构域和5.8S-ITS区域获得的高序列相似性表明，每对物种应被视为一个单一物种。这项工作的主要目的是为大量具有生物技术和临床意义的酵母物种建立一个数据库，并便于对它们进行简单、快速和可靠的鉴定。本研究用属于念珠菌属的75个物种的图谱改进了IATA网页（http://motor.edinfo.es/iata/）上的在线数据库。
