9种发热伴出疹病原体基因芯片检测方法的建立  

Development of a DNA-based microarray for detection of nine pathogens causing rash and fever illness

作　　者：徐胜平[1,2] 刘琪琦[2] 张严峻[3] 王升启[1,2] XU Sheng-ping LIU Qi-qi ZHANG Yan-jun WANG Sheng-qi

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]浙江省疾病预防控制中心,杭州310051

出　　处：《军事医学》2017年第2期135-140,159,共7页Military Medical Sciences

基　　金：国家科技重大专项传染病应急处置检测技术平台资助项目(2011ZX10004-001)

摘　　要：目的建立一种能同时、快速、准确地检测包括:麻疹病毒、风疹病毒、肠道病毒71型、水痘带状疱疹病毒、登革热病毒、人类小DNA病毒B19、柯萨奇病毒A16型、A组β型酿脓链球菌(溶血性链球菌)、伤寒沙门菌,9种发热伴出疹病原体的化学发光基因芯片的方法。方法根据9种病原体特异基因中的高度保守区序列设计引物与探针,进行多重不对称PCR扩增,将带有标记的扩增产物与带有特异性探针的芯片杂交,经洗涤、化学发光显色后进行结果分析。经多重PCR体系、杂交反应和化学发光检测条件的优化,评价该芯片的特异性、灵敏度和重复性。实时荧光PCR法与芯片法分别检测肠道病毒EV71梯度稀释的核酸,比较两种方法的灵敏度。结果共筛选出9对特异性扩增引物和11条特异性检测探针。该芯片具有良好的特异性、灵敏度和重复性。质粒参考品和体外转录RNA参考品的最低检测限为3×10~3拷贝/反应,芯片法的灵敏度为实时荧光PCR法的1/10。检测74例临床样本的灵敏度为95%,特异性为85.7%,总符合率为93.2%。结论建立了一种可同时检测9种发热伴出疹病原体的化学发光基因芯片的检测方法,为发热伴出疹的临床诊断和流行病学调查提供了一种高通量的筛查手段。Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71,Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-β Streptococcus pyogenes（hemolytic streptococcus）and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3x10^3 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95%and 85.7%respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

关 键 词：发热伴出疹 多重PCR 基因芯片 化学发光法 

分 类 号：R440[医药卫生—诊断学] R511[医药卫生—临床医学]