牛布鲁氏菌病、结核、炭疽、口蹄疫、病毒性腹泻黏膜病、副流感、传染性鼻气管炎可视化基因芯片检测方法的建立

Establishment of Visible Gene Chip Detection Method for Brucella,Anthrax,Bovine Tuberculosis,Foot-and-Mouth Disease,Bovine Viral Diarrhoea,Bovine Parainfluenza,Bovine Infectious Rhinotacheitus

针对现有牛病病原检测方法指标单一,操作复杂的现状,拟建立一种可高效检测牛布鲁氏菌病、结核、炭疽、口蹄疫、病毒性腹泻黏膜病、副流感、传染性鼻气管炎的基因芯片技术。根据已公布的各病原核酸序列,设计引物和探针。利用多重PCR方法扩增目的片段,扩增产物与探针特异性杂交,芯片反应显色后肉眼观察进行检测结果判定。优化反应条件、建立检测方法,同时对检测方法的灵敏度、可重复性、特异性、保存期等进行评价。结果显示,该基因芯片检测方法单一病原灵敏度检测可达1.0×10^(-6) ng/μL,混合病原灵敏度检测可达1.4×10^(-5) ng/μL;各病原间无交叉反应,检测健康牛血清、组织,牛流行性热病毒也均无响应;针对同一阳性质控品,芯片重复率达100%。保存期试验表明,芯片在2～8℃至少可保存6个月。检测30份临床样本,结果与标准方法结果一致。实验建立的方法具有高通量、高灵敏度、高特异性等特点,可在3 h内完成同时对七种牛重要疫病的检测,在牛群疫病诊断、净化及流行病学调查等方面有良好的应用前景。

In view of the fact that the bovine pathogen detecting method has a single detection index and a complicated operation, this study is intended to establish a visible gene chip detection method for simultaneous detection of Brucella, Anthrax, Bovine tuberculosis, Foot-and-Mouth disease, Bovine viral diarrhoea, Bovine parainfluenza, Bovine infectious rhinotacheitus. Primers and probes were designed based on the published nucleic acid sequences of each pathogen. The multiplex PCR method was used to amplify the target fragment, and the amplified product was specifically hybridized with the probe. After the color reaction on chip, the result was determined by visual observation. The detection method was further optimized before establishment, and the sensitivity, repeatability, specificity, and shelf life were assessed. The chip detection method was of high sensitivity, good repeatability and specificity. When detecting the single pathogen, the sensitivity can reach 1.0×10^-6 ng/μL. The seven mixed pathogens can reach 1.4×10^-5 ng/μL. There is no cross-reaction among the seven pathogens, the healthy bovine samples, and bovine epizootic fever samples. For the same samples, the same batch and different batches of chips have consistent results. The chip shelf life test shows that the chip can be stored for 6 months at 2~8 °C. The 30 clinical samples test results were consistent with the results of the standard detection methods. The method established in this study is a high-throughput method with high specificity and high sensitivity, which can simultaneously detect the seven important diseases in 3 hours, and can be potentially applied in diagnosis and epidemiological investigation of bovine diseases.