都柏林念珠菌的PCR-RFLP鉴定

目的建立一种准确、可靠的鉴定都柏林念珠菌基因型的方法。方法临床念珠菌分离自临床生殖器念珠菌病患者,45℃温度试验时几乎不生长,且其他表型实验结果也符合都柏林念珠菌特征。对41例临床念珠菌和1例白念珠菌标准株、1例都柏林念珠菌标准株rDNA内部转录间隔区的基因进行聚合酶链反应（PCR）扩增,HpyF10Ⅵ酶切后观察PAGE图谱。结果聚合酶链反应-限制性片段长度多态性（PCR-RFLP）后,39例临床株鉴定为白念珠菌。2例临床菌株带型特殊,测序后行BLAST比对分析,1例鉴定为白念珠菌,另1例尚不能肯定为都柏林念珠菌,还需要进一步以其他分子生物学方法鉴定。结论PCR-RFLP方法酶切后两种念珠菌带型区分明显,可以鉴别大部分临床菌株。基因测序是该方法有意义的补充。

Identification of Candida dubliniensis with PCR-RFLP analysis

Objective To provide a PCR-RFLP method for unequivocal differentiation of C. dubliniensis from C. albicans. Methods All 41 clinical isolates were sampled from patients in the dermatology department of Tianjin General Hospital. Standard isolates of C. albicans （ ATCC-11006 ） and C. dublinniensis （ CBS-8501 ） were presented by Peking University. All clinical isolates were identified using phenotypic tests and grew poorly at 45℃ on SDA. The PCR primers Ⅰ and Ⅱbinded to conserved regions of the Internal Transcribed Spacer regions ITS1 or ITS2 ,respectively. PCR products were digested for 14h at 37℃ with HpyF10VI. Digestion were run on 8% PAGE. Results Thirty-nine clinical isolates were confirmed to be C. albicans , which showed the same RFLP pattern as standard isolates of C. albicans . The other 2 clinical isolates show different RFLP pattens. After sequencing, one was confirmed to be C. albicans , the other maybe was C. dublinniensis. Conclusion Our PCR-RFLP method for two Candida species discrimination have been established. Sequencing is a helpful supplement.