腺病毒分型基因芯片方法的建立和验证

发布时间：2018-04-02 08:02

本文选题：腺病毒科　切入点：分型　出处：《军事医学》2017年02期

【摘要】：目的建立一种高通量、快速、准确甄别3、7、14、11和55型腺病毒的化学发光基因芯片方法。方法从GenBank中查找这5种腺病毒特异性基因保守区,设计分型的探针并在筛选后进行合成,用于制备寡核苷酸基因芯片。运用多重不对称PCR法扩增特异性基因片段,将扩增产物与基因芯片上的特异性探针杂交,经过洗涤、化学发光显色后,进行结果分析。在优化的多重PCR体系、杂交条件和化学发光检测条件下,对芯片的特异性、灵敏性和重复性进行评价。结果建立的基因芯片方法具有良好的特异性、灵敏性和重复性,检测质粒参考品最低检测限为3×10~3拷贝/反应,38例临床样本的分型结果与测序法结果基本一致(37/38)。结论建立了一种能快速、灵敏、特异区分5种腺病毒型别的基因芯片分型方法,为腺病毒分型提供一种新的检测手段。
[Abstract]:Objective to establish a high throughput, rapid and accurate chemiluminescence gene chip method for identifying adenovirus type 3, 7, 1411 and 55. Methods the specific conserved regions of these five adenovirus genes were found from GenBank, the probe for typing was designed and synthesized after screening. Oligonucleotide gene chip was prepared. Specific gene fragments were amplified by multiple asymmetric PCR method. The products were hybridized with specific probes on the gene chip. After washing and chemiluminescence reaction, The results were analyzed. The specificity, sensitivity and repeatability of the microarray were evaluated under the optimized multiplex PCR system, hybridization conditions and chemiluminescence detection conditions. Sensitivity and reproducibility. The minimum detection limit of plasmid reference was 3 脳 10 ~ (-3) copy / reaction. The typing results of 38 clinical samples were in good agreement with the results of sequencing method. Conclusion A rapid and sensitive method was established. The gene chip typing method which specifically distinguishes 5 adenovirus types provides a new detection method for adenovirus typing.
【作者单位】：河北北方学院;军事医学科学院放射与辐射医学研究所;解放军309医院;
【基金】：国家科技重大专项“重大新药创制”资助项目(2015ZX09J15105-001-001)
【分类号】：R440;R511

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