本文關(guān)鍵詞： 非洲豬瘟病毒 基礎(chǔ)RPA 實(shí)時(shí)熒光RPA 側(cè)流層析試紙條RPA　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)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(yán)重的經(jīng)濟(jì)損失。由于ASFV的抗感染機(jī)制極為復(fù)雜,基因型多,至今尚無(wú)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(yù)防。針對(duì)該現(xiàn)狀,本研究成功建立了 ASFV重組酶聚合酶擴(kuò)增技術(shù)(RPA)基礎(chǔ)檢測(cè)方法和重組酶聚合酶擴(kuò)增技術(shù)(RPA)探針法檢測(cè)方法,并從靈敏度、特異性、重復(fù)性、檢測(cè)時(shí)間、檢測(cè)溫度等多方面進(jìn)行分析,證實(shí)了基于RPA技術(shù)的核酸擴(kuò)增方法具有敏感性強(qiáng)、特異性高、反應(yīng)快速、恒溫等特點(diǎn)。為建立重組酶聚合酶擴(kuò)增技術(shù)(RPA)基礎(chǔ)檢測(cè)方法,根據(jù)GcnBank中ASFV基因序列FR682468,針對(duì)該序列p72基因區(qū)域設(shè)計(jì)三組基礎(chǔ)RPA引物,優(yōu)化反應(yīng)時(shí)間、反應(yīng)溫度,進(jìn)行靈敏度、特異性、穩(wěn)定性試驗(yàn),并檢測(cè)樣品。結(jié)果顯示,本方法最佳反應(yīng)條件為39℃C擴(kuò)增20 min。靈敏度試驗(yàn)最低可檢測(cè)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(wú)交叉反應(yīng)。結(jié)果表明,建立的重組酶聚合酶擴(kuò)增技術(shù)(RPA)基礎(chǔ)檢測(cè)方法可快速、靈敏、特異的檢測(cè)ASFV。為建立重組酶聚合酶擴(kuò)增技術(shù)(RPA)探針檢測(cè)方法,本研究針對(duì)ASFV B646L(p72)基因設(shè)計(jì)了3組引物和探針,并進(jìn)行篩選、反應(yīng)條件優(yōu)化、靈敏性、特異性和重復(fù)性試驗(yàn)。結(jié)果顯示,實(shí)時(shí)熒光RPA方法可在39℃C、20 min內(nèi)即可檢測(cè)10個(gè)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(wú)交叉反應(yīng),檢測(cè)5.5× 106-5.5× 100拷貝/μ L共7個(gè)稀釋度樣品在各時(shí)間點(diǎn)的熒光強(qiáng)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(cè)到5.5×101拷貝/μL濃度的質(zhì)粒。目前,該等溫、快速擴(kuò)增方法可用于ASFV的定性檢測(cè),為我國(guó)ASFV感染的早期診斷提供技術(shù)支持,對(duì)疫情爆發(fā)后相應(yīng)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(hào)：1552398