变性梯度胶电泳对结膜炎细菌多态性分析  

16S rDNA-based analysis of bacterial diversity of conjunctitis by DGGE and sequence

作　　者：兰和魁[1] 陈丽珊[2] 马晓晓[1] 雷艳[1] 吴艺[1] 任大明[2] 

机构地区：[1]南方医科大学珠江医院,广州510282 [2]复旦大学遗传所,上海200433

出　　处：《发育医学电子杂志》2013年第3期129-135,共7页Journal of Developmental Medicine (Electronic Version)

摘　　要：目的应用聚合酶链反应-变性梯度胶电泳(PCR-DGGE)研究泪液细菌多态性和致病病原体。方法直接从泪液标本中抽取总DNA,扩增16S rDNA V3区,DGGE分离后测序与传统培养方法进行比较,并作出相应的评价。结果检测131份标本,其中59份化脓性结膜炎标本54份呈阳性结果,39份非化脓性结膜炎标本15份为阳性,而34份正常泪液标本6例扩增出目的片段。检测细菌种类依次为:葡萄球菌、棒状杆菌、链球菌、不动杆菌、枯草杆菌、假单胞菌、丙酸菌属及泛菌属。有3个序列在属的水平上不能被确定,其序列分别与肠细菌、不经培养的真细菌及不经培养的β蛋白细菌有高度的序列相似性。培养方法只对葡萄状球菌、棒状杆菌、链球菌、假单胞菌,以及克雷伯氏杆菌属呈阳性。结论 PCR-DGGE印迹技术和16S rDNA序列分析能快速识别细菌性结膜炎患者中的病原菌,而且是检测和鉴定那些不能用传统培养法检测的单种细菌或多种细菌眼部感染的较为适宜的方法。Objectives To study bacterial diversity and pathogen in ocular environment by PCR-DGGE and sequencing. Methods A new molecular biology technique for identifying multiple bacteria from the ocular environment developed in this paper. From 131 human conjunctivae(58 with purulent, 39 with nonpurulent conjunctivitis and 34 normal control), swabs were taken and DNAs were extracted. V3 region of the 16S rDNA was amplified by polymerase chain reaction(PCR) and separated by denaturing gradient gel electrophoresis(DGGE). Then DGGE bands were excised and directly sequenced, or cloned to pGEM-T vector and sequenced. Variable segments of each sequence were compared with the sequences in GenBank. Furthermore, the results were compared with those obtained from the conventional cultivation. Results Targeted fragments could be amplified from 54 of 59 cases in purulent conjunctiovitis eyes, from 15 of 39 in nonpurulent conjunctivitis eyes and from 6 of 34 in normal controls. The following genera ordinal were detected: Staphylococcus, Corynebacterium, Streptococcus, Acinetobacter, Bacillus,Pseudomonas, Propionibacterium, and pantoea. Three sequences could not be identified to genus level. The sequences had the highest similarity to enterobacteria, uncultured eubacterium and uncultured beta proteobacterium. Cultures were only positive for Staphylococcus, Corynebacterium, Streptococcus, Pseudomonas, and Klebsiella oxytoca. Some bacteria need commensal culture or co-culture.Conclusion Our data suggest that combination of 16S rDNA sequence analysis and DGGE fingerprinting is a rapid alternative to identify pathogens in patients with bacterial conjunctivitis and an appropriate method for detection and identification of monomicrobial/polymicrobial ocular infection which might not be detected by conventional cultivation.

关 键 词：变性梯度胶电泳 16S RDNA 结膜炎 细菌多态性 

分 类 号：R777.31[医药卫生—眼科]