口蹄疫—水疱性口炎—猪水疱病联合共检基因芯片的构建

发布时间：2018-06-10 20:33

  本文选题：口蹄疫病毒 + 水疱性口炎病毒　； 参考：《四川农业大学》2015年硕士论文

【摘要】：口蹄疫、猪水疱病和水疱性口炎分别是由口蹄疫病毒(Foot-and-mouth disease virus,FMDV)、猪水疱病病毒(Swine vesicular disease virus,SVDV)和水疱性口炎病毒(Vesicular stomatitis virus,VSV)引起的急性动物传染病,都可以感染猪,且由于发病症状相似,从发病症状上鉴别难度较大,因此构建一种能同时鉴别诊断三种疾病和区分口蹄疫三种血清型的高通量联合检测方法具有重要意义。本研究以FMDV O型、A型、Asia 1型、VSV和SVDV为研究对象,建立了寡核苷酸基因芯片检测体系。研究内容如下：1.共检基因芯片的靶基因引物和探针的构建：参考GenBank中已公布的基因组序列,根据基因芯片引物设计要求,选择三种病毒的保守区域设计引物和探针,对引物和探针进行了验证,构建了基于口蹄疫病毒的A、O、Asia 1型VP1序列、VSV的L基因和SVDV VP1序列的pMD19-T克隆质粒并测序鉴定。结果显示,克隆的靶基因与NCBI公布的序列一致性均在95%以上。2.共检基因芯片的构建和优化设计了基因芯片矩阵和位置指控、杂交质控及杂交质控探针序列,并对三种病毒的探针进行了筛选。对芯片点样后的水化温度与封闭条件、PCR退火温度和循环数、荧光基团标记引物的使用量、杂交温度等进行了优化。结果表明：使用醛基基片,在18℃-37℃水化过夜,使用含0.25% BH4Na,25%乙醇的封闭液进行封闭所制的芯片效果较好；PCR扩增时选择58℃退火温度,一般使用2μL荧光基团标记引物,扩增30个循环便可得到足够的产物；在临床检测时为提高灵敏度可使用4-8μL荧光基团标记引物,扩增40-50个循环,杂交温度达到42℃便可获得有意义的结果,为保证特异性,在52℃进行杂交效果更好。3.共检基因芯片的效果评价对所构建的基因芯片检测方法进行了灵敏性试验、特异性试验、重复性试验和稳定性试验。结果表明,本研究所设计的FMDV-O引物PCR检测限为0.1180ng/mL, FMDV-A为0.0184ng/mL, FMDV-Asia I为0.129ng/mL, VSV为0.0267ng/mL, SVDV为0.0247ng/mL,芯片检测方法灵敏度至少比PCR检测高一个数量级；特异性良好,所设计引物没有出现非特异性扩增,能准确区分检测出FMDV、VSV、SVDV,并能区分出口蹄疫病毒的三种血清型；应用了两家公司所生产的不同批次醛基基片进行重复性验证,均能够较好地实现重复实验；将点制好的芯片抽真空后于4℃保存,至少能保存四个月。
[Abstract]:Foot-and-mouth disease (FMD), porcine blisters disease (BMD) and vesicular stomatitis (BMD) are acute animal infections caused by foot-and-mouth disease virus (Foot-and-mouth disease virus), Swine vesicular disease virus (SVDVV), and vesicular stomatitis virus (Vesicular stomatitis virus VSVV), all of which can infect pigs and have similar symptoms. It is difficult to identify the symptoms of the disease, so it is of great significance to establish a high throughput combined detection method which can simultaneously differentiate and diagnose the three diseases and distinguish the three serotypes of foot-and-mouth disease (FMD). In this study, the detection system of oligonucleotide microarray was established using VSV and SVDV of FMDV O and Asia 1. The research is as follows: 1. Construction of target gene primers and probes for co-detection of gene chips: according to the genomic sequence published in GenBank and according to the design requirements of gene chip primers, the primers and probes of three kinds of viruses were selected to design, and the primers and probes were verified. The L gene of VSV and pMD19-T of VP1 sequence of SVDV based on foot-and-mouth disease virus (FMDV) were constructed and sequenced. The results showed that the identity of the cloned target gene with the NCBI published sequence was more than 95%. The construction and optimization of the gene chip were used to construct and optimize the gene chip matrix, position control, hybridization quality control and hybridization quality control probe sequences, and the probes of three viruses were screened. The hydration temperature, PCR annealing temperature and cycle number, the amount of fluorescent group labeled primers and hybridization temperature were optimized. The results showed that the chip made by using aldehyde substrate, hydrating overnight at 18 鈩，

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