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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2008, Vol. 28 Issue (专刊): 1-6    
论文     
中枢神经系统感染性疾病病原体检测芯片的初步研究
庞靖祥 韩金祥 高雪芹 潘继红
山东省医药生物技术研究中心 山东省医学科学院生物技术研究中心 山东省医药生物技术研究中心 山东省医药生物技术研究中心
Development of Oligonucleotide microarray for the detection of pathogen of central nervous system infectious disease and its application
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摘要:

目的:构建一种用于检测巨细胞病毒(CMV)、风疹病毒(RV)、单纯疱疹病毒I型(HSVI)、结核分枝杆菌(TB)、EB病毒(EBV)五种病原体的寡核苷酸芯片,用于中枢神经系统疾病的常见病原体的病因学诊断,为临床治疗提供依据。方法:首先根据病原体的特异性蛋白的相关序列设计70 mer寡核苷酸长探针,在优化探针的基础上,选择特异性好的探针构建不同种属多种病原体的寡核苷酸芯片,用随机引物方法对靶基因进行生物素直接标记,标记产物与芯片杂交后再与avidin-cy3反应,对杂交阵列洗涤后扫描。采用该寡核苷酸芯片对临床样本进行检测并与和PCR检测方法进行检测比较。结果:通过标准菌株检测证明该检测芯片具有很高的特异性。对临床标本的检测结果表明,我们研制的寡核苷酸芯片检测体系与PCR法的检测结果相比,两种检测方法没有显著性差异( >0.05),且一致性较好(Kappa值均大于0.75),其特异度均在95%左右,敏感度均在83%以上。但是我们研制的寡核苷酸芯片只需要一次检测就可以完成对5种常见病原体的检测。结论:本实验构建的寡核苷酸芯片检测体系具有快速、方便、准确的特点,可以高通量的检测临床样本而无须进行细菌培养,为中枢神经系统重要病原体检测提供了一种有效工具。

Abstract:

To construct a oligonucleotide microarray for the detection of five important pathogens including CMV、RV、HSVI、TB、EBV in central nervous system..METHODS : 70 mer oligo probes targeted the specific protein of 5 common pathogens of infectious diseases of the nervous system were designed for the fabrication of microarray. DNA was extracted with conmercilized kit. The target genes were labeled with biotin-dUTP by random primer extension method using the DNA as template. The labeled products were hybridized with the oligonucleotide microarray. After hubridization the microarray were binding with avidin-CY3. After scan with Scanarray 4000 confocal scanner, the fluorescent intensity was analyzed with Quantarray3.0 software. This oligonucelotide microarray system were used to detect clinical sample and compared with PCR method. RESULT: The specific probes for the pathogens are very specific for the detecting the presence of the pathogens. Clinical detection results showed the specificity and sensitivity of this microarray system are 95% and 83% respectively. There is no significant difference between our method and the routine PCR method.. CONCLUSION:The microaaray system for the detection of the major pathogen of central nervous system has the advantage of specific,sensitive and parallel detection. We can add pathogen probes according to the practical needs. It can not only be used for the diagnosis of central nervous system, but also canbe used for the detection of blood, urine, and other body fluid infection.

收稿日期: 2008-01-17 出版日期: 1900-01-01
通讯作者: 韩金祥   
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引用本文:

庞靖祥,韩金祥,高雪芹,潘继红. 中枢神经系统感染性疾病病原体检测芯片的初步研究[J]. 中国生物工程杂志, 2008, 28(专刊): 1-6.

. Development of Oligonucleotide microarray for the detection of pathogen of central nervous system infectious disease and its application. China Biotechnology, 2008, 28(专刊): 1-6.

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https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I专刊/1

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