【摘要】 目的用限制性片段长度多态性聚合酶链反应(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术快速准确鉴定外阴阴道念珠菌病(VVC)相关念珠菌菌种,并对科玛嘉显色培养法、Vitek 2 YST鉴定卡和PCR-RFLP 3种方法鉴定念珠菌菌种的效果进行方法学评价。方法收集贵阳市妇幼保健院VVC患者感染的念珠菌菌种100株,用十六烷基三甲基溴化铵法(cetyltrimethylammonium bromide, CTAB)法提取念珠菌DNA,PCR扩增念珠菌DNA的ITS片段并进行测序分析确定念珠菌菌种;分别采用科玛嘉显色培养法、Vitek 2 YST鉴定卡和PCR-RFLP 3种方法对其进行鉴定,以测序结果为"金标准",比较3种方法鉴定念珠菌菌种的正确率。结果科玛嘉显色培养法中5株葡萄牙念珠菌与2株酿酒念珠菌显色错误,均显淡紫色;Vitek 2 YST鉴定卡中,1株白念珠菌与2株热带念珠菌鉴定不出,Cyberlindnera fabianii、Candida orthopsilosis与Candida metapsilosis鉴定错误;PCR-RFLP采用内切酶MspⅠ可成功鉴定念珠菌中除Candida metapsilosis、Candida orthopsilosis与近平滑念珠菌之外的其他菌种,进一步采用内切酶ApaⅠ与NcoⅠ可将Candida metapsilosis、Candida Orthopsilosis与近平滑念珠菌三者鉴别开。科玛嘉显色培养法对白念珠菌、克柔念珠菌、热带念珠菌等3种念珠菌鉴定正确率100%,对光滑念珠菌的鉴定正确率为73.1%;Vitek 2 YST鉴定卡可鉴定常见念珠菌且正确率较高,不能鉴定Cyberlindnera fabianii、Candida orthopsilosis与Candida metapsilosis等非常见念珠菌;PCR-RFLP技术可快速准确鉴定所有念珠菌菌种。结论 PCR-RFLP技术为早期准确鉴定临床念珠菌感染提供了新的选择,具有很好的应用前景。更多还原

【Abstract】 Objective To identify rapidly and accurately the vulvovaginal candidosis(VVC)-related Candida species by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technology, and compare the results of CHROMagar chromogenic method, Vitek 2 YST identification card and PCR-RFLP to evaluate its methodological quality. Methods A total of 100 strains of Candida species were collected from VVC infected subjects in Guiyang Maternal and Child Health-Care Hospital. Candida DNA was extracted by cetyltrimethylammonium bromide(CTAB) method. The ITS fragments of Candida DNA were amplified and sequenced, and the sequence was considered as the "gold standard" for the detection. The accuracy, sensitivity and specificity of three methods, i.e., CHROMagar chromogenic method, Vitek 2 YST identification card and PCR-RFLP, in the identification for Candida species were compared. Results In the results of identification by CHROMagar chromogenic method, 5 strains of Portuguese yeasts and 2 strains of Saccharomyces cerevisiae showed wrong lilacy colour. By Vitek 2 YST identification card, 1 strain of Candida albicans and 2 strains of Candida tropicalis could not be identified and the identification results of Cyberlindnera fabianii, Candida orthopsilosis and Candida metapsilosis were wrong. By using endonuclease MspⅠ enzyme PCR-RFLP method could successfully identify all the Candida species except Candida albicans, Candida orthopsilosis and Candida metapsilosis which could be differentiated further by using endonuclease ApaⅠ and NcoⅠ. The accuracy rates of identification results by CHROMagar chromogenic method was all 100% for Candida albicans, Candida krusei and Candida tropicalis, but low specificity(73.1%) for Candida glabrata. Vitek 2 YST identification card could identify common Candida with high accuracy, but could not identify the rare candida, such as Cyberlindnera fabianii, Candida orthopsilis and Candida metapsilosis. PCR-RFLP technique could identify all Candida species quickly and accurately. Conclusion PCR-RFLP technique may provide a new option for early and accurate identification of clinical Candida infection, which should have good application prospect.更多还原