牛羊疫病多重PCR及多重?zé)晒舛縋CR檢測(cè)方法的建立
發(fā)布時(shí)間：2018-08-02 07:54
【摘要】：近年來(lái),國(guó)家大力發(fā)展草食畜牧業(yè),牛羊養(yǎng)殖數(shù)量不斷增加,規(guī)模化程度不斷提升。隨著牛羊養(yǎng)殖的迅猛發(fā)展,牛羊疫病的流行形式趨于復(fù)雜,牛羊疫病常呈現(xiàn)發(fā)病增多的趨勢(shì),通過(guò)對(duì)單一病種的實(shí)驗(yàn)室檢測(cè)很難作出確診。因此,建立可以同時(shí)分別對(duì)多種疫病進(jìn)行快速鑒別診斷的多重PCR和多重?zé)晒舛縋CR,對(duì)牛羊疫病的實(shí)驗(yàn)室快速檢測(cè)尤為重要,對(duì)基層動(dòng)物疫病防控有很強(qiáng)的實(shí)際應(yīng)用價(jià)值。本研究以小反芻獸疫(Pestedes petits ruminants,PPR)、羊痘(Capripox,CP)、藍(lán)舌病(Bluetongue,BT)、牛病毒性腹瀉(Bovine Viral Diarrhea,BVD)和牛傳染性鼻氣管炎(Infectious bovine rhinotracheitis,IBR)等牛羊常發(fā)病為研究對(duì)象。通過(guò)大量比對(duì)GenBank中PPRV的N基因、CPV的P32基因和BTV的NS3基因,選取高度保守的基因序列作為靶序列,建立了 PPRV、BTV、CPV的單重PCR方法,同時(shí)還建立了單管同時(shí)擴(kuò)增PPRV、BTV、CPV的多重PCR方法。通過(guò)大量比對(duì)GenBank中BVDV的5' UTR基因和IBRV的gB基因,選取高度保守的基因序列作為靶序列,建立了 BVDV、IBRV的單重PCR方法,同時(shí)建立了單管同時(shí)擴(kuò)增BVDV、IBRV的雙重PCR方法。結(jié)果表明,建立的單重PCR方法具有較好的敏感性和特異性,PPRV的最低檢測(cè)限為60 pg,BTV的最低檢測(cè)限為1 pg,CPV的最低檢測(cè)限為8 pg,BVDV的最低檢測(cè)限為0.12 ng,IBRV的最低檢測(cè)限為0.6 pg。建立的PPRV、BTV、CPV多重PCR方法也具有良好的敏感性和特異性,PPRV的最低檢測(cè)限為4 pg,BTV為10 pg,CPV為0.8 pg,建立的BVDV、IBRV多重PCR方法BVDV最低檢測(cè)限為0.12 ng,IBRV為6 pg。在常規(guī)PCR基礎(chǔ)上,通過(guò)設(shè)計(jì)引物和探針建立了 PPRV、BTV、CPV的單重?zé)晒舛縋CR方法,同時(shí)還建立了單管同時(shí)擴(kuò)增PPRV、BTV、CPV的多重?zé)晒舛糠椒?以及BVDV、IBRV的單重?zé)晒舛縋CR方法和單管同時(shí)擴(kuò)增BVDV、IBRV的雙重?zé)晒舛縋CR方法。結(jié)果表明,建立的單重?zé)晒舛糠椒ň哂休^好的敏感性和特異性,PPRV的最低檢測(cè)限為0.06 pg,BTV的最低檢測(cè)限為0.1 pg,CPV的最低檢測(cè)限為0.04 pg,BVDV的最低檢測(cè)限為O.1pg,IBRV的最低檢測(cè)限為0.06 pg。建立的PPRV、BTV、CPV多重?zé)晒舛糠椒ㄒ簿哂辛己玫拿舾行院吞禺愋?PPRV的最低檢測(cè)限為0.06 pg,BTV為0.12pg,CPV為0.04 pg,建立的BVDV、IBRV多重?zé)晒舛縋CR方法BVDV最低檢測(cè)限為1.2 pg,IBRV 為 0.8 pg�？傊�,本研究建立的PPRV、BTV、CPV單重、多重PCR方法和單重、多重?zé)晒舛縋CR方法和BVDV、IBRV單重、多重PCR方法和單重、多重?zé)晒舛縋CR方法,能夠準(zhǔn)確的對(duì)這幾種牛羊常見(jiàn)病進(jìn)行快速的檢測(cè),具有較強(qiáng)的應(yīng)用價(jià)值。
[Abstract]:In recent years, the state has made great efforts to develop herbivorous animal husbandry, the quantity of cattle and sheep breeding has been increasing and the scale of the animal husbandry has been increasing. With the rapid development of cattle and sheep breeding, the epidemic form of cattle and sheep epidemic disease tends to be complex, and the epidemic disease of cattle and sheep often presents a trend of increasing incidence, so it is difficult to make a diagnosis by laboratory testing of single disease. At the same time, multiple PCR and multiple fluorescence quantitative PCR were used for rapid differential diagnosis of multiple diseases. It was very important for laboratory rapid detection of cattle and sheep blight, and it was of great practical value for prevention and control of primary animal diseases. This study was based on Pestedes petits ruminants (PPR), sheep pox (Capripox, CP), blue tongue disease (Bluetongue, B). T), cattle and sheep, such as bovine viral diarrhea (Bovine Viral Diarrhea, BVD) and bovine infectious nasotracheitis (Infectious bovine rhinotracheitis, IBR), are often studied. Single weight PCR method and multiple PCR methods for simultaneous amplification of PPRV, BTV and CPV by single tube were also established. A highly conservative gene sequence was selected as the target sequence by a large number of BVDV 5'UTR genes and IBRV gB genes in GenBank. The results show that the proposed single PCR method has good sensitivity and specificity, the minimum detection limit of PPRV is 60 PG, the minimum detection limit of BTV is 1 PG, the minimum detection limit of CPV is 8 PG, the minimum detection limit of BVDV is 0.12 ng, and the minimum detection limit of IBRV is 0.6 PG. PPRV. The minimum detection limit of PPRV is 4 PG, BTV is 10 PG, CPV is 0.8 PG, BVDV, IBRV multiple PCR method BVDV minimum detection limit is 0.12 ng, IBRV is 6. Light quantitative method, single heavy fluorescence PCR method of BVDV and IBRV and single tube amplification of BVDV and IBRV double fluorescence quantitative PCR method. The results show that the established single heavy fluorescence quantitative method has good sensitivity and specificity, the minimum detection limit of PPRV is 0.06 PG, the minimum detection limit of BTV is 0.1 PG, and the lowest detection limit of CPV is 0.04 P. The minimum detection limit of G, BVDV is O.1pg, the minimum detection limit of IBRV is 0.06 pg., PPRV, BTV, CPV multiple fluorescence quantitative methods also have good sensitivity and specificity, the minimum detection limit of PPRV is 0.06 PG, BTV is 0.12pg, and the minimum detection limit is 1.2. In a word, g., PPRV, BTV, CPV single weight, multiple PCR method and single weight, multiple fluorescence quantitative PCR method and BVDV, IBRV single weight, multiple PCR method and single weight, multiple fluorescent quantitative PCR, can accurately detect the common diseases of these cattle and sheep, and have strong application value.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S854.4
，
本文編號(hào)：2158721
[Abstract]:In recent years, the state has made great efforts to develop herbivorous animal husbandry, the quantity of cattle and sheep breeding has been increasing and the scale of the animal husbandry has been increasing. With the rapid development of cattle and sheep breeding, the epidemic form of cattle and sheep epidemic disease tends to be complex, and the epidemic disease of cattle and sheep often presents a trend of increasing incidence, so it is difficult to make a diagnosis by laboratory testing of single disease. At the same time, multiple PCR and multiple fluorescence quantitative PCR were used for rapid differential diagnosis of multiple diseases. It was very important for laboratory rapid detection of cattle and sheep blight, and it was of great practical value for prevention and control of primary animal diseases. This study was based on Pestedes petits ruminants (PPR), sheep pox (Capripox, CP), blue tongue disease (Bluetongue, B). T), cattle and sheep, such as bovine viral diarrhea (Bovine Viral Diarrhea, BVD) and bovine infectious nasotracheitis (Infectious bovine rhinotracheitis, IBR), are often studied. Single weight PCR method and multiple PCR methods for simultaneous amplification of PPRV, BTV and CPV by single tube were also established. A highly conservative gene sequence was selected as the target sequence by a large number of BVDV 5'UTR genes and IBRV gB genes in GenBank. The results show that the proposed single PCR method has good sensitivity and specificity, the minimum detection limit of PPRV is 60 PG, the minimum detection limit of BTV is 1 PG, the minimum detection limit of CPV is 8 PG, the minimum detection limit of BVDV is 0.12 ng, and the minimum detection limit of IBRV is 0.6 PG. PPRV. The minimum detection limit of PPRV is 4 PG, BTV is 10 PG, CPV is 0.8 PG, BVDV, IBRV multiple PCR method BVDV minimum detection limit is 0.12 ng, IBRV is 6. Light quantitative method, single heavy fluorescence PCR method of BVDV and IBRV and single tube amplification of BVDV and IBRV double fluorescence quantitative PCR method. The results show that the established single heavy fluorescence quantitative method has good sensitivity and specificity, the minimum detection limit of PPRV is 0.06 PG, the minimum detection limit of BTV is 0.1 PG, and the lowest detection limit of CPV is 0.04 P. The minimum detection limit of G, BVDV is O.1pg, the minimum detection limit of IBRV is 0.06 pg., PPRV, BTV, CPV multiple fluorescence quantitative methods also have good sensitivity and specificity, the minimum detection limit of PPRV is 0.06 PG, BTV is 0.12pg, and the minimum detection limit is 1.2. In a word, g., PPRV, BTV, CPV single weight, multiple PCR method and single weight, multiple fluorescence quantitative PCR method and BVDV, IBRV single weight, multiple PCR method and single weight, multiple fluorescent quantitative PCR, can accurately detect the common diseases of these cattle and sheep, and have strong application value.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S854.4
，
本文編號(hào)：2158721
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