牛羊疫病多重PCR及多重荧光定量PCR检测方法的建立
发布时间：2018-08-02 07:54
【摘要】：近年来,国家大力发展草食畜牧业,牛羊养殖数量不断增加,规模化程度不断提升。随着牛羊养殖的迅猛发展,牛羊疫病的流行形式趋于复杂,牛羊疫病常呈现发病增多的趋势,通过对单一病种的实验室检测很难作出确诊。因此,建立可以同时分别对多种疫病进行快速鉴别诊断的多重PCR和多重荧光定量PCR,对牛羊疫病的实验室快速检测尤为重要,对基层动物疫病防控有很强的实际应用价值。本研究以小反刍兽疫(Pestedes petits ruminants,PPR)、羊痘(Capripox,CP)、蓝舌病(Bluetongue,BT)、牛病毒性腹泻(Bovine Viral Diarrhea,BVD)和牛传染性鼻气管炎(Infectious bovine rhinotracheitis,IBR)等牛羊常发病为研究对象。通过大量比对GenBank中PPRV的N基因、CPV的P32基因和BTV的NS3基因,选取高度保守的基因序列作为靶序列,建立了 PPRV、BTV、CPV的单重PCR方法,同时还建立了单管同时扩增PPRV、BTV、CPV的多重PCR方法。通过大量比对GenBank中BVDV的5' UTR基因和IBRV的gB基因,选取高度保守的基因序列作为靶序列,建立了 BVDV、IBRV的单重PCR方法,同时建立了单管同时扩增BVDV、IBRV的双重PCR方法。结果表明,建立的单重PCR方法具有较好的敏感性和特异性,PPRV的最低检测限为60 pg,BTV的最低检测限为1 pg,CPV的最低检测限为8 pg,BVDV的最低检测限为0.12 ng,IBRV的最低检测限为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异性,PPRV的最低检测限为4 pg,BTV为10 pg,CPV为0.8 pg,建立的BVDV、IBRV多重PCR方法BVDV最低检测限为0.12 ng,IBRV为6 pg。在常规PCR基础上,通过设计引物和探针建立了 PPRV、BTV、CPV的单重荧光定量PCR方法,同时还建立了单管同时扩增PPRV、BTV、CPV的多重荧光定量方法,以及BVDV、IBRV的单重荧光定量PCR方法和单管同时扩增BVDV、IBRV的双重荧光定量PCR方法。结果表明,建立的单重荧光定量方法具有较好的敏感性和特异性,PPRV的最低检测限为0.06 pg,BTV的最低检测限为0.1 pg,CPV的最低检测限为0.04 pg,BVDV的最低检测限为O.1pg,IBRV的最低检测限为0.06 pg。建立的PPRV、BTV、CPV多重荧光定量方法也具有良好的敏感性和特异性,PPRV的最低检测限为0.06 pg,BTV为0.12pg,CPV为0.04 pg,建立的BVDV、IBRV多重荧光定量PCR方法BVDV最低检测限为1.2 pg,IBRV 为 0.8 pg。总之,本研究建立的PPRV、BTV、CPV单重、多重PCR方法和单重、多重荧光定量PCR方法和BVDV、IBRV单重、多重PCR方法和单重、多重荧光定量PCR方法,能够准确的对这几种牛羊常见病进行快速的检测,具有较强的应用价值。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S854.4
，
本文编号：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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S854.4
，
本文编号：2158721
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