改良PCR-RFLP方法快速鉴别常见致病念珠菌  

Rapid diagnosis of six medically important Candida species with modified PCR-RFLP method

作　　者：王淼淼[1,2] 张晓利[3] 吕雪莲 蔡晴[1] 葛一平[1] 沈永年[1] 吕桂霞[1] 刘维达[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,江苏南京210042 [2]苏州大学第一附属医院皮肤科,江苏苏州215006 [3]暨南大学临床医学博士后流动站,广东广州510632 [4]大连皮肤病医院,辽宁大连116021

出　　处：《临床皮肤科杂志》2013年第11期646-648,共3页Journal of Clinical Dermatology

基　　金：科技重大专项传染病领域念珠菌侵袭性感染早期诊断技术平台的建立(2013ZX1004612-005)资助项目

摘　　要：目的:建立快速准确鉴别常见致病念珠菌菌落的PCR-限制性内切酶片段长度多态性(RFLP)方法。方法:结合直接菌落PCR检测技术,使用通用引物扩增6种念珠菌(包括白念珠菌、克柔念珠菌、近平滑念珠菌、光滑念珠菌、热带念珠菌和季也蒙念珠菌)ITS1-ITS2区。分别用HhaⅠ、HaeⅢ、HinfⅠ、MspⅠ和Hpy F10Ⅵ等5种内切酶对PCR产物进行酶切,建立以菌落PCR为基础的RFLP方法,然后对30株临床株进行PCR-RFLP图谱分析。结果:应用MspⅠ进行酶切反应,6种念珠菌的酶切带型有明显差异,易于鉴别。结论:通过直接菌落PCR检测技术,使用MspⅠ进行酶切,可以经1次反应,简便快速鉴别出常见的6种致病念珠菌,为临床念珠菌感染的快速准确诊断赢得时间。Objective： To developed a PCR-RFLP method for diagnosis and differentiation of common pathogenic Candida species rapidly and accurately. Methods： In combination with PCR directly on yeast colonies, universal fungal primers were used to amplify the ITS region of Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Candida guilliermondii, followed by restriction fragment length polymorphism （RFLP） analysis with the enzymes Hha I, Hae III, Hinf I, Msp I and HpyF10VI. 30 clinical isolates were identified with the developed PCR-RFLP method. Results： The six medically important Candida species could be distinguished on the basis of the sequence of the ITS region using Msp I restriction enzyme reaction. Conclusions： The six common pathogenic Candida species can be rapidly and simply identified with PCR directly on colonies in combination with Msp I restriction enzyme reaction. This technique is fast and accurate for clinical diagnosis of Candida species.

关 键 词：念珠菌 菌落PCR 限制性内切酶片段长度多态性 

分 类 号：R756.5[医药卫生—皮肤病学与性病学]