液相核酸芯片鉴定角膜常见致病真菌的初步实验研究  

Preliminary study on the use of DNA suspension array for the identification of corneal pathogenic fungi

作　　者：董燕玲[1] 谢立信[1] 银红梅[1] 孙士营[1] 黄海南[2] 

机构地区：[1]山东省眼科研究所山东省眼科学重点实验室-省部共建国家重点实验室培育基地,青岛266071 [2]山东省医药生物技术研究中心

出　　处：《中华眼科杂志》2009年第2期110-114,共5页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（30630063,30271394）;山东省科技攻关计划项目（2004GG2202154）

摘　　要：目的探讨建立快速鉴定角膜常见致病真菌的液相核酸芯片检测系统及其应用于真菌性角膜炎临床快速诊断的可行性。方法实验研究。设计针对5种角膜常见致病真菌（茄病镰刀菌、串珠镰刀菌、尖孢镰刀菌、烟曲霉菌及黄曲霉菌）rRNA基因内转录间隔（ITS）区的种特异性探针，5’端氨基C12修饰后与不同荧光色的微球结合，建立液相核酸芯片检测系统。将目的真菌的5株标准菌株和42株角膜分离保存菌株的基因组DNA以一对真菌通用引物扩增并标记后用于杂交检测。实验中设市单一菌种榆测与多菌种混合检测的比较，以及芯片检测与琼脂糖凝胶电泳检测的比较，采用配对设计资料的t检验和Spearman等级相关分析对检测结果进行统计学分析，进而评估该检测系统的特异性、灵敏度和重复性。结果所建市的液相核酸芯片体系可在聚合酶链反应（PCR）后3h内准确将5株标准菌株鉴定至菌种的水平；42株角膜分离保存菌株单次检测阳性率达95．2％；阳件信号与背景比值位于5．6～13．3之间；单一菌种检测和5种菌种平行检测的中位荧光强度（MFI）差异无统计学意义（t=0．2524，P=0．8132）；MFI的变化与PCR产物的量呈正相关（rs=1．0000，P〈0．01）；最低检测浓度为0．94ngPCR产物，比琼脂糖凝胶电泳低40倍；4次重复检测的变异系数在1．8％-13．7％之间。结论所建立的液相芯片检测系统具有较高的特异性、灵敏度及重复性，可同时完成对5种角膜常见致病真菌的菌种检测，为液相芯片技术在临床真菌快速诊断中的应用奠定了实验基础。Objective To develop a multiplex, microsphere-based DNA suspension array for the identification of important pathogenic fungi of cornea and to study the feasibility of its application in the clinical diagnosis of fungal keratitis. Methods Fusarium solani, fusarium moniliforme, fusarium oxysporum, aspergillus fumigatus and fspergillus flavus, which covered about 80% of pathogenic fungi of fungal keratitis, were chosen as target species of this study. Five species-specific capture probes were designed in the internal transcribed spacer （ITS） regions of ribosomal DNA and synthesized with 5＇ amino modifier C12 to covalently bind to different sets of fluorescent beads. Biotinylated amplicons of 5 reference strains and 42 clinical strains were generated with a pair of universal primers to yield fragments for detection. Comparison between single species detection and multiplex detection were designed, as well as detection between array and agarose gel electrophoresis （AGE）. Spearman rank correlation analysis and t test were applied to evaluate the specificity, sensibility and reproducibility of suspension array. Results Five reference strains and 40 of 42 （95.2%） clinical strains were correctly identified within 3 h post-PCR amplification while 2 other clinical strains were not identified because of their high background fluorescence intensity. Positive S/B ranged from 5.6 to 13.3. There was no significant difference between respective detection and mixed detection of 5 species （t = 0. 2524, P = 0. 8132）. The sensitivity limit for this assay was determined to be 0. 94 ng PCR products. The MFI presented positive correlation with amount of PCR products （rs = 1. 0000, P 〈 0. 01 ）. Coefficient variation of four repeated detections was 1.8%-13.7%. Conclusion The suspension array is a rapid, sensitive and specific method for the identification of the most important species of corneal pathogenic fungi and might be used in the clinical laboratories.

关 键 词：寡核苷酸序列分析 角膜 真菌 

分 类 号：R686[医药卫生—骨科学]