CAJ | 학술논문

目的应用基因芯片对临床常见的8种致病细菌进行检测.方法选取8种临床常见的致病细菌,包括金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、大肠杆菌、奇异变形杆菌、产气肠杆菌、荧光假单胞菌、宋内志贺菌.以16S rRNA基因为目的基因,白行设计通用引物系列扩增目的片段,针对高变区域设计探针,建立基因芯片检测体系,并对所选细菌进行检测.收集来自武汉大学中南医院的待检标本50份,包括血液6份、痰液32份、粪便9份、纤维支气管镜检测物3份,提取DNA后利用建立的基因芯片进行检测.结果自行设计的通用引物能很好地扩增目的片段,针对8种细菌进行多批次重复检测,结果显示所选用的探针具有很好的检测效果.对临床50份标本进行检测,其中47份得到准确的检测结果,3份未得到结果的原因为芯片未包含菌种.通过优化检测过程,可在8h内报告检测结果.探针信号随时间的衰减程度观察表明,60d后探针信号虽有衰减,但其衰减程度对检测结果判断无影响.结论本研究设计的基因芯片检测体系能准确而快速地对临床常见细菌做出检测,为基因芯片技术应用于临床奠定基础.
목적 응용기인심편대림상상견적8충치병세균진행검측.방법 선취8충림상상견적치병세균,포괄금황색포도구균、동록가단포균、폐염극뢰백균、대장간균、기이변형간균、산기장간균、형광가단포균、송내지하균.이16S rRNA기인위목적기인,백행설계통용인물계렬확증목적편단,침대고변구역설계탐침,건립기인심편검측체계,병대소선세균진행검측.수집래자무한대학중남의원적대검표본50빈,포괄혈액6빈、담액32빈、분편9빈、섬유지기관경검측물3빈,제취DNA후이용건립적기인심편진행검측.결과 자행설계적통용인물능흔호지확증목적편단,침대8충세균진행다비차중복검측,결과현시소선용적탐침구유흔호적검측효과.대림상50빈표본진행검측,기중47빈득도준학적검측결과,3빈미득도결과적원인위심편미포함균충.통과우화검측과정,가재8h내보고검측결과.탐침신호수시간적쇠감정도관찰표명,60d후탐침신호수유쇠감,단기쇠감정도대검측결과판단무영향.결론 본연구설계적기인심편검측체계능준학이쾌속지대림상상견세균주출검측,위기인심편기술응용우림상전정기출.
Objective To detect eight kinds of clinical common pathogenic bacteria by DNA microarray.Methods Eight kinds of common pathogenic bacteria,including Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis,Enterobacter aerogenes,Pseudomonas fluorescens,Shigella sonnei were collected.Universal primers were designed to amplify 16S rRNA gene fragment from the genomic DNA of the eight bacteria,and probes were designed in the highly variable regions.DNA microarray detection system was established and used for detection of colleted bacteria.A total of 50 samples were collected from the Zhongnan Hospital of Wuhan University,including 6 blood samples,32 sputum samples,9 feces samples and 3 bronchoscope lavage samples.DNA were extracted and detected by the established DNA microarray system.Results The desired fragments were well amplified by the self-designed universal primers.The selected probes had good detection results according to repeated detection.Of the 50 samples detected,pathgenic bacteria were accurately detected in 47 samples.Other three samples were not detected as those bacteria were not included in the chip.By optimizing the detection process,the results could be reported within 8 hours.Observation of probe signal attenuation indicated that even attenuated after 60 days,but the attenuation did not affect the results.Conclusion A microarray system was established for detection of clinical common bacteria accurately and quickly,which provided foundation for its clinical application.