非洲猪瘟病毒重组酶聚合酶扩增（RPA）方法的建立

发布时间：2018-03-01 15:00

  本文关键词： 非洲猪瘟病毒 基础RPA 实时荧光RPA 侧流层析试纸条RPA　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的一种猪急性、热性、高度接触性传染病,给养猪业造成了严重的经济损失。由于ASFV的抗感染机制极为复杂,基因型多,至今尚无有效疫苗用于防控,阻止该病爆发主要依赖于早期快速诊断和预防。针对该现状,本研究成功建立了 ASFV重组酶聚合酶扩增技术(RPA)基础检测方法和重组酶聚合酶扩增技术(RPA)探针法检测方法,并从灵敏度、特异性、重复性、检测时间、检测温度等多方面进行分析,证实了基于RPA技术的核酸扩增方法具有敏感性强、特异性高、反应快速、恒温等特点。为建立重组酶聚合酶扩增技术(RPA)基础检测方法,根据GcnBank中ASFV基因序列FR682468,针对该序列p72基因区域设计三组基础RPA引物,优化反应时间、反应温度,进行灵敏度、特异性、稳定性试验,并检测样品。结果显示,本方法最佳反应条件为39℃C扩增20 min。灵敏度试验最低可检测到5.5 × 101拷贝/u L,与经典PCR方法灵敏度相同,与猪瘟病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪传染性胃肠炎病毒无交叉反应。结果表明,建立的重组酶聚合酶扩增技术(RPA)基础检测方法可快速、灵敏、特异的检测ASFV。为建立重组酶聚合酶扩增技术(RPA)探针检测方法,本研究针对ASFV B646L(p72)基因设计了3组引物和探针,并进行筛选、反应条件优化、灵敏性、特异性和重复性试验。结果显示,实时荧光RPA方法可在39℃C、20 min内即可检测10个拷贝的DNA分子,且与猪瘟病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪传染性胃肠炎病毒无交叉反应,检测5.5× 106-5.5× 100拷贝/μ L共7个稀释度样品在各时间点的荧光强度,变异系数范围在0.38%-28.30%。侧流层析试纸条RPA最低可检测到5.5×101拷贝/μL浓度的质粒。目前,该等温、快速扩增方法可用于ASFV的定性检测,为我国ASFV感染的早期诊断提供技术支持,对疫情爆发后相应控制方案的制定具有重要意义。
[Abstract]:African swine swine virus (ASFV) is a kind of acute, hot and highly contact infectious disease caused by African swine fever virus (ASFV), which has caused serious economic losses to the pig industry. Because of the complexity of anti-infection mechanism of ASFV, it has many genotypes. So far, there is no effective vaccine for prevention and control, and preventing the outbreak mainly depends on early rapid diagnosis and prevention. In this study, the basic detection method of ASFV recombinant enzyme polymerase amplification technique and the detection method of recombinant enzyme polymerase amplification technique were successfully established, and the sensitivity, specificity, repeatability and detection time were analyzed. It was proved that the nucleic acid amplification method based on RPA technology had the characteristics of high sensitivity, high specificity, fast reaction, constant temperature and so on. According to the ASFV gene sequence FR682468 in GcnBank, three groups of basic RPA primers were designed for the region of p72 gene of the sequence. The reaction time, reaction temperature, sensitivity, specificity, stability were optimized, and the samples were tested. The optimum reaction conditions were as follows: amplification at 39 鈩，

本文编号：1552398