本文關(guān)鍵詞： 非洲豬瘟病毒 基礎RPA 實時熒光RPA 側(cè)流層析試紙條RPA　出處：《內(nèi)蒙古農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：非洲豬瘟(African swine fever,ASF)是由非洲豬瘟病毒(African swine fever virus,ASFV)感染引起的一種豬急性、熱性、高度接觸性傳染病,給養(yǎng)豬業(yè)造成了嚴重的經(jīng)濟損失。由于ASFV的抗感染機制極為復雜,基因型多,至今尚無有效疫苗用于防控,阻止該病爆發(fā)主要依賴于早期快速診斷和預防。針對該現(xiàn)狀,本研究成功建立了 ASFV重組酶聚合酶擴增技術(shù)(RPA)基礎檢測方法和重組酶聚合酶擴增技術(shù)(RPA)探針法檢測方法,并從靈敏度、特異性、重復性、檢測時間、檢測溫度等多方面進行分析,證實了基于RPA技術(shù)的核酸擴增方法具有敏感性強、特異性高、反應快速、恒溫等特點。為建立重組酶聚合酶擴增技術(shù)(RPA)基礎檢測方法,根據(jù)GcnBank中ASFV基因序列FR682468,針對該序列p72基因區(qū)域設計三組基礎RPA引物,優(yōu)化反應時間、反應溫度,進行靈敏度、特異性、穩(wěn)定性試驗,并檢測樣品。結(jié)果顯示,本方法最佳反應條件為39℃C擴增20 min。靈敏度試驗最低可檢測到5.5 × 101拷貝/u L,與經(jīng)典PCR方法靈敏度相同,與豬瘟病毒、豬圓環(huán)病毒2型、豬流行性腹瀉病毒、豬傳染性胃腸炎病毒無交叉反應。結(jié)果表明,建立的重組酶聚合酶擴增技術(shù)(RPA)基礎檢測方法可快速、靈敏、特異的檢測ASFV。為建立重組酶聚合酶擴增技術(shù)(RPA)探針檢測方法,本研究針對ASFV B646L(p72)基因設計了3組引物和探針,并進行篩選、反應條件優(yōu)化、靈敏性、特異性和重復性試驗。結(jié)果顯示,實時熒光RPA方法可在39℃C、20 min內(nèi)即可檢測10個拷貝的DNA分子,且與豬瘟病毒、豬圓環(huán)病毒2型、豬流行性腹瀉病毒、豬傳染性胃腸炎病毒無交叉反應,檢測5.5× 106-5.5× 100拷貝/μ L共7個稀釋度樣品在各時間點的熒光強度,變異系數(shù)范圍在0.38%-28.30%。側(cè)流層析試紙條RPA最低可檢測到5.5×101拷貝/μL濃度的質(zhì)粒。目前,該等溫、快速擴增方法可用于ASFV的定性檢測,為我國ASFV感染的早期診斷提供技術(shù)支持,對疫情爆發(fā)后相應控制方案的制定具有重要意義。
[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，

本文編號：1552398