4种病原菌特异基因片段阳性蜱的16SrRNA基因克隆文库分析  

16S rRNA gene clone library analysis of bacterial communities of the tick with infection of 4 species of pathogens

作　　者：张守印[1] 孙继民[1] 贺金荣[1] 付秀萍[1] 张景山[1] 张建华[1] 蔡虹[1] 马凤琴[1] 海荣[1] 俞东征[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所人兽共患病室(传染病预防控制国家重点实验室),北京102206

出　　处：《中国地方病学杂志》2009年第3期294-297,共4页Chinese Jouranl of Endemiology

基　　金：基金项目：国家科技攻关计划课题（2003BA712A02）

摘　　要：目的建立16SrRNA基因克隆文库分析蜱媒菌群的方法，观察该方法对蜱寄生病原菌的检测效率以及对细菌群落多样性分析和对病原菌的筛检能力。方法用伯氏疏螺旋体、汉赛巴通体、嗜吞噬细胞无形体和查菲埃立克体4种病原菌特异基因设计引物，扩增蜱标本提取的核酸，以上述4种病原菌特异基因片段扩增均阳性的蜱核酸提取物做模板，用16SrRNA基因的通用引物进行PCR扩增、纯化、连接、克隆和测序，建立16SrRNA基因克隆文库，将测序结果与数据库进行比对，计算克隆文库Coverage值和Shannon—Wiener多样性指数。结果测出103个有效序列，检出16种已知种属的细菌，其中8个是优势类型（克隆子数〉5个）；检测到伯氏疏螺旋体、汉赛巴通体和立克次体3种病原菌，但这3种病原菌均不是优势类型（克隆子数均〈5个）。Coverage值为96．11％，Shannon—Wiener多样性指数为2．40。克隆序列分析结果表明，蜱寄生细菌主要为仅、1变形菌纲，占56．25％（9／16）。结论16SrRNA基因序列分析可以对蜱标本进行菌群相对定量研究，可以同时检出多种病原菌．是一种较好的细菌菌群多样性分析和病原菌筛检方法。Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, BartoneUa henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus- or species-bacteria were detected in 103 valid sequences. Eight species were edge type （Clone No. 〉 5 ）. Three kinds of pathogens were identified （Borrelia burgdorferi, Bartonella henselae and Rickettsia sp ）. Three kinds of pathogens were not edge type（Clone No. 〈 5）. Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% （9/16）. Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It＇s a good method for detection of pathogens and bacterial flora analysis.

关 键 词：蜱 16S RRNA基因 序列分析 多样性 

分 类 号：R686[医药卫生—骨科学]