脑脊液病原菌16S rRNA微型寡核苷酸芯片检测方法的初步研究  

ESTABLISHMENT OF 16S rRNA OLIGO-CHIPS TO DETECT PATHOGENS IN CEREBROSPINAL FLUID

作　　者：毕春霞[1] 武静 张德坤[1] 苏维奇[1] 刘成玉[3] 闫志勇[4] 

机构地区：[1]青岛市市立医院检验科,山东青岛266011 [2]海军青岛保障基地医院 [3]青岛大学医学院临床检验诊断学教研室 [4]青岛大学医学院微生物学教研室

出　　处：《青岛大学医学院学报》2006年第3期200-202,共3页Acta Academiae Medicinae Qingdao Universitatis

基　　金：山东省卫生厅青年基金(JW10)

摘　　要：目的初步建立一种脑脊液标本中常见病原菌的快速检测方法。方法根据脑脊液标本中常见病原菌16S rRNA基因保守区的序列设计用于基因扩增的通用引物,制备细菌的通用探针、革兰阳性和阴性菌通用探针,以及肠杆菌科属、嗜血杆菌属和脑膜炎奈瑟菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌、肺炎链球菌、产单核李斯特菌的属或种特异性探针,将探针加尾后固定于尼龙膜上制成寡核苷酸芯片。提取标本中病原菌DNA后用通用引物行PCR扩增,扩增过程中用生物素标记;将产物变性后分别与点有各种探针的尼龙膜反向杂交,检测脑脊液中细菌的感染情况。结果所有探针均只与其相应的细菌DNA扩增产物反应产生杂交信号。结论建立的脑脊夜病原菌寡核苷酸芯片能够准确、敏感、快速、高效地检测脑脊液标本中病原菌的感染情况。Objective To develop a rapid detection method for common pathogens in cerebrospinal fluid （CSF）. Methods According to the conservative and variable regions in bacterial 16S rRNA genes, a pair of universal primers that can amplify rDNA of all bacteria were designed. Ten kinds of specific probes were designed by Premier software. Probes for identification were tailed and then spotted onto a nylon membrane. DNA were isolated from each pathogen, and subjected to UP PCR to amplify tar get fragments, which were labeled withbio 16-dUTPat the same time. All these fragments were denatured, and then hybridized to all probes on nylon membrane and visualized by AKP labeled avidin. Results The pathogens reacted only to their corresponding probe fixed on nylon membrane with high specificity. Conclusion The newly established 16S rRNA oligo chip can specifically screen out the pathogens in CSF.

关 键 词：脑脊髓液 细菌 寡核苷酸芯片 寡核苷酸序列分析 

分 类 号：R450[医药卫生—治疗学]