16SrDNA生物芯片微阵列方法检测立克次体的初步研究  

Development of a universal microarray system for the detection of rickettsiae

作　　者：崔红[1] 陈霞萍[2] 陈梅玲[1] 郭兆彪[1] 王津[1] 翟俊辉[1] 杨瑞馥[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071 [2]湖北石堰市太和医院,石堰442000

出　　处：《军事医学科学院院刊》2003年第3期186-188,222,共4页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的 :以 1 6SrDNA为对象 ,生物芯片微阵列技术为平台 ,建立一种能在未知标本中快速检测 6种立克次体的方法。方法 :针对每种立克次体 1 6SrDNA的可变区 ,分别设计和合成 4条寡核苷酸探针。利用微阵列技术构建立克次体检测用生物芯片。待测立克次体染色体DNA用 1 6SrRNA通用引物扩增掺入荧光素 ,然后与芯片杂交。利用各种立克次体的 4条特异性探针是否全部出现杂交信号来做出判断。结果 :以寡核苷酸为探针的生物芯片系统可以实现 6种立克次体的检测。对伯氏考克斯体、汉氏巴尔通体、恙虫病东方体 ,本系统可以将其鉴定到种或属的位置。从第 4号探针的不同可以将普氏立克次体和立氏立克次体这两个群区分开来。犬埃立克体检测始终为阴性 ,可能和没有合适的标本有关。从接到样品到判读出结果 ,整个检测过程大约需要 4 .5h。敏感性检测结果表明本法比PCR_电泳法敏感 1 0倍。结论 :利用 1 6SrDNA生物芯片微阵列方法可以快速检测Objective:To develop a microarray_based detection system which employs 16S rDNA sequence as its detection target for rapid and efficient detection of rickettsiae. Methods:Specific probes targeting 6 species of rickettsiae were designed by using some bioinformatics softwares and methods.Ten strains of Rickettsiae were tested by using this microarray system.Results:The results showed that Bartonella henselae , Orientia tsutsugamushi , Rickettsiarickettsii , R.prowazekii , Coxiella burnetii could be detected at species or genus level. For example, R.rickettsii had a cross reaction with 3 out of 4 probes specifically targeting R.prowazekii , while R.prowazekii hybridized with only 2 R.rickettsii′s probes. Ehrlichia canis was negative throughout the whole experiment and the reason was under evaluation. The sensitivity assay was performed by employing serial dilution of C.burnetii chromosomal DNA.The sensitivity of detection system used was found to be 10 times higher than that of PCR_electrophoresis. Conclusion: The oligonucleotide microarray could determine most of the test strains at species level. The overall time for sample process, hybridization and data acquisition lasts about 4.5?hours. The oligonucleotide microarray can be used for the detection of rickettsiae.

关 键 词：立克次体 16S RDNA 生物芯片 微阵列 检测 

分 类 号：R376[医药卫生—病原生物学]