可视化基因芯片技术检测肠道病毒方法的建立  

Visual DNA microarrays for simultaneous detection of ten enteroviruses

作　　者：彭贤慧[1] 刘琪琦[2] 陈苏红[2] 刘志红[2] 张敏丽[2] 宋宏彬[3] 王升启[2] 

机构地区：[1]解放军总医院,北京100853 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]军事医学科学院疾病预防控制所,北京100071

出　　处：《军事医学》2012年第4期299-303,共5页Military Medical Sciences

基　　金：广东省教育部产学研结合项目(2011A090200071)

摘　　要：目的建立一种能同时检测10种肠道病毒的可视化基因芯片法。方法根据公开发表的10种常见肠道病毒的序列,设计病毒引物和探针,制备肠道病毒检测基因芯片。利用多重不对称PCR法扩增样品中的病毒靶片段,标记产物与基因芯片上的探针杂交,经清洗、可视化显色后进行结果分析。在优化的RT-PCR体系、杂交反应和可视化检测条件下,评价芯片的特异性、灵敏度和重复性。结果本研究共筛选出2对通用引物、3对特异性引物和1条肠道病毒属通用探针、9条特异性检测探针。该芯片具有较好的特异性和重复性,可检测出不低于102拷贝/μl的体外转录RNA。30例临床标本的芯片检测结果与荧光PCR法一致。结论本研究所建立的方法具有高通量、高特异性、高灵敏度等特点,因此在临床上具有潜在的应用前景,可以为肠道病毒诊断提供实验室依据。Objective To develop a visual DNA microarray for simultaneous detection of ten enteroviruses.Methods Primers and probes were designed based on the conserved sequences of available enterovirus genomes.The probes were immobilized on glass surface to prepare DNA microarrays.The nucleic acids of the selected viruses were amplified and labeled by multiplex asymmetric PCR method,and then hybridized with the microarray.The microarray was scanned after washing and visualized coloration,and the results were analyzed.Facilitated by the optimization of the RT-PCR system,hybridization,and visualization,we evaluated the specificity,sensitivity and reproducibility of the chip.Results Two universal primers,three specific primers one universal probe,and nine specific probes were selected,and the microarray demonstrated upstanding sensitivity and specificity,which could detect no less than 102 copies/μl in vitro transcribed RNA.Thirty stool specimens were tested by this chip.The results were identical to those shown in the assays of fluorescence real-time PCR.Conclusion Our results demonstrate that the visual DNA microarray for detection of ten enteroviruses is of high throughput,specificity and sensitivity.It has the potential in clinical applications.

关 键 词：肠道病毒 基因芯片 多重不对称PCR 可视化 

分 类 号：R373.25[医药卫生—病原生物学]