虫媒病病原多重液相芯片检测方法的建立
发布时间：2018-11-22 16:42
【摘要】：目的：虫媒病是指以昆虫作为媒介通过吸血传播造成人畜感染的一类疾病。本研究利用液相芯片技术建立虫媒病多重病原的检测方法。方法：根据蓝舌病病毒(BTV)、鹿流行性出血热病毒(EHDV)、Q热贝氏柯克斯体(Coxiella burnetii)、非洲猪瘟病毒(ASFV)、西尼罗病毒(WNV)、莱姆病伯氏疏螺旋体(BB)、水泡性口炎病毒(VSV)、裂谷热病毒(RVFV)、埃博拉病毒(EBV)和施马伦贝格病毒(SBV)的高度保守区域分别设计了10对引物与10种探针。选用不同编码的磁性微球分别与10种虫媒病的特异性探针进行有效偶联。在此基础上,通过上下游引物比例、液相杂交温度、液相杂交时间等关键因素的优化,建立了10种虫媒病原的多重液相芯片检测体系。随后对该多重液相体系的特异性、灵敏度和重复性进行验证。结果：通过PCR/RT-PCR扩增得到的片段与预期大小相吻合。将纯化后的PCR产物与载体连接构建阳性质粒,经过DNAMAN分析比对,其同源性均大于97%。当引物比例为1：2,杂交温度为50℃,杂交时间为30min时,每种虫媒病的荧光中位值明显高于背景值,能有效、特异的检测出目的片段。结果显示整个体系对口蹄疫、小反刍兽疫等疫病无交叉反应,最小检出量是102拷贝数并且具有良好的重复性。利用建立的多重液相芯片体系对已知虫媒病原混合样品和300份蚊、蜱、蠓的未知核酸进行检测,发现病原均为阳性且内蒙古呼伦贝尔6只蜱中携带伯氏疏螺旋体。结论：本研究成功建立了能同时检测十种虫媒病病原的液相芯片方法,为多种虫媒病原高通量快速筛查搭建了技术平台,这对动物虫媒性疫病的防控以及鉴定等都将具有重要意义。
[Abstract]:Objective: insecticidal disease is a kind of disease which is transmitted by sucking blood by insects. In this study, a liquid-phase microarray technique was used to detect multiple pathogens of insect-borne diseases. Methods: according to bluetongue virus (BTV), deer epidemic hemorrhagic fever virus (EHDV), Q Rebekovskii virus (Coxiella burnetii), African classical swine fever (ASFV), West Nile virus (WNV), Lyme disease spirochetes (BB), 10 pairs of primers and 10 probes were designed for highly conserved regions of vesicular stomatitis virus (VSV),) Rift Valley Fever virus (RVFV),) Ebola virus (EBV) and Schmalenberger virus (SBV), respectively. Magnetic microspheres with different codes were effectively coupled with 10 specific probes of insect-borne diseases. On this basis, the multiplex liquid-phase microarray detection system of 10 kinds of insect-borne pathogens was established by optimizing the key factors such as the proportion of primers, the temperature of liquid phase hybridization and the time of liquid phase hybridization. The specificity, sensitivity and repeatability of the multiplex liquid system were then verified. Results: the fragments amplified by PCR/RT-PCR coincided with the expected size. The purified PCR product was ligated with the vector to construct the positive plasmid. The homology of the purified PCR product was more than 97 by DNAMAN analysis. When the primer ratio was 1: 2, the hybridization temperature was 50 鈩，
本文编号：2349919
[Abstract]:Objective: insecticidal disease is a kind of disease which is transmitted by sucking blood by insects. In this study, a liquid-phase microarray technique was used to detect multiple pathogens of insect-borne diseases. Methods: according to bluetongue virus (BTV), deer epidemic hemorrhagic fever virus (EHDV), Q Rebekovskii virus (Coxiella burnetii), African classical swine fever (ASFV), West Nile virus (WNV), Lyme disease spirochetes (BB), 10 pairs of primers and 10 probes were designed for highly conserved regions of vesicular stomatitis virus (VSV),) Rift Valley Fever virus (RVFV),) Ebola virus (EBV) and Schmalenberger virus (SBV), respectively. Magnetic microspheres with different codes were effectively coupled with 10 specific probes of insect-borne diseases. On this basis, the multiplex liquid-phase microarray detection system of 10 kinds of insect-borne pathogens was established by optimizing the key factors such as the proportion of primers, the temperature of liquid phase hybridization and the time of liquid phase hybridization. The specificity, sensitivity and repeatability of the multiplex liquid system were then verified. Results: the fragments amplified by PCR/RT-PCR coincided with the expected size. The purified PCR product was ligated with the vector to construct the positive plasmid. The homology of the purified PCR product was more than 97 by DNAMAN analysis. When the primer ratio was 1: 2, the hybridization temperature was 50 鈩，
本文编号：2349919
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2349919.html
