基因芯片在小儿败血症病原诊断中应用的初步研究-儿科学专业论文.docxVIP

基因芯片技术在小儿败血症病原诊断中应用的初步研究中文摘要 基因芯片技术在小儿败血症病原诊断中应用的初步研究 中文摘要 败血症是严重威胁小儿生命的疾病之一，目前的诊断手段包括血培养等都存 在很大的不足，因此需要一种快速、敏感、准确的诊断方法。本研究主要是借鉴PCR 和基因芯片技术的原理，设计和制作败血症病原的简易诊断芯片，并探讨在此过程 中的一系列技术问题，包括探针的设计、固定、芯片的杂交条件、杂交信号的检测 以及待检标本中靶基因的扩增。 我们以10种引起败血症最常见的细菌为实验材料。芯片检测的靶基因是 16SrRNA基因，利用它的保守性设计通用引物，利用它的特异性设计各菌种的特异 性探针。共设计了13个探针，通过加尾和加热固定制成简易芯片后和lO个菌种的 16SrRNA基因扩增产物杂交，实验过程中确定最优化的杂交条件。 随后将确立的方法应用于临床标本。我们选取55例在入院时被诊断为败血症 的患儿。结果检出19份阳性，其中表皮葡萄球菌15份，大肠杆菌2份，肺炎克雷 伯菌l份。血培养有9份阳性，其中有8份标本检出的菌种和芯片检测结果完全符 合。血培养的阳性率为16．3％，而芯片检测为34．5％，P0．05，两者差异有显著意 义，以血培养为金标准则芯片检测的敏感性为88．9％，特异性76．2％。 本研究证实了PCR结合基因芯片检测病原菌方法的可行性。PCR扩增技术使 本方法的敏感性大大高于血培养。芯片的技术特点确保检测的特异性。因此我们认 为PCR结合基因芯片技术在败血症病原检测中是一种有前途的方法。 关键词：基因芯片PCR 16SrRNA 败血症 Basic Basic study of genechip technique oil pathogen diagnosis of children’s bacteremia ABSTRACT Bacteremia is one of the diseases which menace severely the lives of neonatals． There are many defects in diagnostic methods including blood cultivation at the present time．A more rapid，sensitive and accurate assay is highly demanded．The main purpose of this research is to design and set up a kind of diagnostic microarray on pathogens of neonatal bacteremh employing the principles ofPCR and genechip．A sedes oftechnique problems Were studied on design and fixation of probes，parameters of hybridization, detection ofhybridization signals and amplification oftarget gene． 10 species of pathogens of bacteremia were taken as experimental materials． 16SrRNA gene Was chosen as the target gene with its conservation employed to design primers for amplification and variation used to design specific probes．13 probes were designed and the genechip Was achieved by tailing and fixation．The amplicons of 16SrRNA gene of 10 pathogens were hybridized with this genechip，thus the optimical hybridization parameters were determined． After all parameters ofhybridization were defined，genechip Was used on 55 clinical blood specimens collected‰patients diagnosed as bacteremia when they Were enrolled into our hospital．The resu