PE、EV71及CoxA16基因探针的制备

发布时间：2018-11-18 11:42

【摘要】：目的制备地高辛标记基因探针,利用分子杂交试验同时检测手足口病患者粪便标本中的肠道病毒(Pan-enterovirus,PE)、肠道病毒71型(Enterovirs 71EV71)和柯萨奇病毒A组16型(Coxsachie virus,CoxA16)。方法根据GenBank已发表的基因序列,分别在PE、EV71、CoxA16保守区设计3对特异性寡核苷酸引物,进行实时荧光定量PCR检测。从PE、EV71、CoxA16阳性标本中分离病毒,提取RNA,以RT-PCR扩增产物作为基因探针的目的基因,采用随机引物法进行Digxigenin-11-dUTP标记,采用分子杂交试验检测3种基因探针的灵敏性和特异性。结果 RTPCR分别扩增出116、226、208bp目的基因片段,经标记后作为检测PE、EV71、CoxA16的3种高纯度的基因探针,对相应病毒RNA的最低检出限均为1pg,可分别与PE(+)、EV71(+)、CoxA16(+)标本提取的RNA发生特异性杂交,与乙型脑炎病毒、诺如病毒、轮状病毒、H3N2、甲型H1N1流感病毒的核酸无杂交反应。结论制备的PE、EV71及CoxA16基因探针灵敏、特异,操作简单,适用于手足口病病原的实验室检测。
[Abstract]:Objective to prepare digoxigenin-labeled gene probe and to detect enterovirus (Pan-enterovirus,PE), enterovirus 71 (Enterovirs 71EV71) and coxsackie virus A group 16 (Coxsachie virus,CoxA16) in fecal specimens of patients with hand, foot and mouth disease by molecular hybridization. Methods according to the published gene sequence of GenBank, three pairs of specific oligonucleotide primers were designed in the conserved region of PE,EV71,CoxA16 and detected by real-time fluorescence quantitative PCR. The virus was isolated from PE,EV71,CoxA16 positive samples, and the target gene of RNA, was extracted with RT-PCR amplification products as gene probe. Random primer method was used for Digxigenin-11-dUTP labeling. The sensitivity and specificity of three gene probes were detected by molecular hybridization test. Results the 116226208bp target gene fragments were amplified by RTPCR and labeled as three high purity gene probes for detection of PE,EV71,CoxA16. The minimum detection limit for the corresponding virus RNA was 1 PG, which could be compared with PE (), EV71 (),. The RNA extracted from CoxA16 () samples showed specific hybridization, and no hybridization reaction was found with Japanese encephalitis virus, norovirus, rotavirus, H _ 3N _ 2 and A H1N1 influenza virus. Conclusion the PE,EV71 and CoxA16 gene probes prepared are sensitive, specific and easy to operate, and can be used for the laboratory detection of the pathogens of hand, foot and mouth disease.
【作者单位】：泰安市疾病预防控制中心;
【基金】：泰安市科学技术发展计划项目(No.201340629)
【分类号】：R440;R725.1

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