CAJ | 학술논문

目的建立16S rRNA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16S rRNA基因的通用引物进行PCR扩增、纯化、连接、克隆和测序,建立16S rRNA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α、γ变形菌纲,占56.25%(9/16).结论 16S rRNA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.
목적 건립16S rRNA기인극륭문고분석비매균군적방법,관찰해방법대비기생병원균적검측효솔이급대세균군락다양성분석화대병원균적사검능력.방법 용백씨소라선체、한새파통체、기탄서세포무형체화사비애립극체4충병원균특이기인설계인물,확증비표본제취적핵산,이상술4충병원균특이기인편단확증균양성적비핵산제취물주모판,용16S rRNA기인적통용인물진행PCR확증、순화、련접、극륭화측서,건립16S rRNA기인극륭문고,장측서결과여수거고진행비대,계산극륭문고Coverage치화Shannon-Wiener다양성지수.결과 측출103개유효서렬,검출16충이지충속적세균,기중8개시우세류형(극륭자수>5개);검측도백씨소라선체、한새파통체화립극차체3충병원균,단저3충병원균균불시우세류형(극륭자수균<5개).Coverage치위96.11%,Shannon-Wiener다양성지수위2.40.극륭서렬분석결과표명,비기생세균주요위α、γ변형균강,점56.25%(9/16).결론 16S rRNA기인서렬분석가이대비표본진행균군상대정량연구,가이동시검출다충병원균,시일충교호적세균균군다양성분석화병원균사검방법.
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, Bartonella 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.