腺病毒分型基因芯片方法的建立和验证  

Dvelopment of a DNA-based microarray for detection of human adenovirus serotypes

作　　者：陈晓飞[1,2] 刘琪琦[2] 周伟[3] 张五星[3] 王升启[2] CHEN Xiao-fei LIU Qi-qi ZHOU Wei ZHANG Wu-xing WANG Sheng-qi(Hebei North University, Zhangjiakou,Hebei 075000, China Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China The 309th Hospital of PLA, Beijing 100091, China)

机构地区：[1]河北北方学院,河北张家口075000 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]解放军309医院,北京100091

出　　处：《军事医学》2017年第2期130-134,共5页Military Medical Sciences

基　　金：国家科技重大专项"重大新药创制"资助项目(2015ZX09J15105-001-001)

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。Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,quick and accurate detection of serotypes of human adenovirus（HAdV）,namely,HAdV3,HAdV7,HAdV11,HAdV14 and HAdV55.Methods Based on the specific gene sequences in the conserved region of adenovirus from GenBank,oligonucleotide primers and probes were designed and synthesized to prepare the oligonucleotide microarray.The specific genomic sequences were amplified by multiplex PCR method.The multiplex PCR amplification products were hybridized with the specific probes of microarray that was scanned after washing and chemiluminescenceb before the result was analyzed.After optimization of the multiple PCR system,hybridization reactions and conditions of chemiluminescence,the specificity,sensitivity and reproducibility of the chip were evaluated.Results The microarray displayed high sensitivity,specificity and reproducibility.The minimum detection limit of plasmidg DNA was 3 ×10^3 copies/reaction.The microarray detection results of 38 clinical samples were approximately consistent with those using the direct sequencing method（37/38）.Conclusion A chemiluminescence imaging DNA microarray method for quick,sensitive and specific detection of five serotypes of HAdV is established,which can provide a new means for detecting serotypes of HAdV.

关 键 词：腺病毒科 分型 基因芯片 化学发光 

分 类 号：R440[医药卫生—诊断学] R511[医药卫生—临床医学]