猪瘟和蓝耳病快速检测方法的建立和应用
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摘要
猪瘟是由黄病毒科瘟病毒属猪瘟病毒引起的猪的一种高度传染性病毒病。猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒引起的一种严重危害养猪业的接触性传染病。近些年来,这两种病的爆发给我国的养猪业带来的巨大的损失。快速、准确、简便的检测方法对于控制和预防这两种病都有重要的意义,本研究从分子生物学和免疫学出发,建立了几种快速检测这两种病毒的方法,这对养猪业的健康发展有着重要的意义。
本研究根据猪瘟病毒和猪繁殖与呼吸综合征病毒的基因保守序列设计了2对针对这2种病毒的特异引物,并建立了多重RT-PCR一步法同时检测这两种病毒。根据Genbank发表的猪瘟病毒5’NTR核苷酸序列设计了一对特异性引物,并建立了SYBR Green I实时荧光定量PCR检测猪瘟病毒方法。根据Genbank发表的繁殖与呼吸综合征病毒M基因核苷酸序列设计了一对特异性引物,并建立了SYBR Green I实时荧光定量PCR检测猪繁殖与呼吸综合征病毒的方法。
本研究根据这两种病毒的免疫学特性,在猪瘟、猪繁殖与呼吸综合征单重斑点免疫金渗滤法的基础上,建立能同时检测猪瘟和猪繁殖与呼吸综合征的双重斑点免疫金渗滤法。
这些方法的建立,不仅对这两种疾病的临床检测提供了理论依据和方法指导,而且对这两种疾病的预防和临床检测方法的进一步研究都具有十分重要的意义。
Hog Cholera(HC) is a highly infectious viral disease caused by the agent of Hog Cholera virus which is the member of genus pestivirus, the family flaviviridae. Porcine reproductive and respiratory syndrome(PRRS), aroused by the PRRS virus, is a kind of highly contagious disease that is a great danger to swine cultivation industry. In recent years,those two diseases were take great side effects to swine industry. Due to establishing rapid,specific and convienent diagnosis technology is critical to control and prevent two diseases, this research using molecular biology and immunology developed few methods for detection of HC and PRRS and it have greatly significance to swine cultivation industry.
According to HC and PRRS virus conservative sequence,this study was designed two pairs of specific HC and PRRS primers,and developed Multiplex RT-PCR which could detect HC and PRRS simultaneously. one pair of specific primer was designed according to the HC virus 5’NTR nucleotide sequence downloaded from Genebank,moreover,established SYBR GreenI fluorescent real-time RT-PCR method for detection of HC virus. one pair of specific primer was designed according to the PRRS M gene nucleotide seqence downloaded from Genebank,moreover,established SYBR GreenI fluorescent real-time RT-PCR method for detection of PRRS virus.
According to the immunology property of HC and PRRS virus, based on HC and PRRS single dot immunogold filtration assay (DIGFA),this study developed the double DIGFA that could detect HC and PRRS simultaneously.
Establishing those methods not only provide the theoretical foundation and method for clinical detection of two diseases,but also is significant to conduct further study for the prevention and clinical detection of two diseases.
本研究根据猪瘟病毒和猪繁殖与呼吸综合征病毒的基因保守序列设计了2对针对这2种病毒的特异引物,并建立了多重RT-PCR一步法同时检测这两种病毒。根据Genbank发表的猪瘟病毒5’NTR核苷酸序列设计了一对特异性引物,并建立了SYBR Green I实时荧光定量PCR检测猪瘟病毒方法。根据Genbank发表的繁殖与呼吸综合征病毒M基因核苷酸序列设计了一对特异性引物,并建立了SYBR Green I实时荧光定量PCR检测猪繁殖与呼吸综合征病毒的方法。
本研究根据这两种病毒的免疫学特性,在猪瘟、猪繁殖与呼吸综合征单重斑点免疫金渗滤法的基础上,建立能同时检测猪瘟和猪繁殖与呼吸综合征的双重斑点免疫金渗滤法。
这些方法的建立,不仅对这两种疾病的临床检测提供了理论依据和方法指导,而且对这两种疾病的预防和临床检测方法的进一步研究都具有十分重要的意义。
Hog Cholera(HC) is a highly infectious viral disease caused by the agent of Hog Cholera virus which is the member of genus pestivirus, the family flaviviridae. Porcine reproductive and respiratory syndrome(PRRS), aroused by the PRRS virus, is a kind of highly contagious disease that is a great danger to swine cultivation industry. In recent years,those two diseases were take great side effects to swine industry. Due to establishing rapid,specific and convienent diagnosis technology is critical to control and prevent two diseases, this research using molecular biology and immunology developed few methods for detection of HC and PRRS and it have greatly significance to swine cultivation industry.
According to HC and PRRS virus conservative sequence,this study was designed two pairs of specific HC and PRRS primers,and developed Multiplex RT-PCR which could detect HC and PRRS simultaneously. one pair of specific primer was designed according to the HC virus 5’NTR nucleotide sequence downloaded from Genebank,moreover,established SYBR GreenI fluorescent real-time RT-PCR method for detection of HC virus. one pair of specific primer was designed according to the PRRS M gene nucleotide seqence downloaded from Genebank,moreover,established SYBR GreenI fluorescent real-time RT-PCR method for detection of PRRS virus.
According to the immunology property of HC and PRRS virus, based on HC and PRRS single dot immunogold filtration assay (DIGFA),this study developed the double DIGFA that could detect HC and PRRS simultaneously.
Establishing those methods not only provide the theoretical foundation and method for clinical detection of two diseases,but also is significant to conduct further study for the prevention and clinical detection of two diseases.
引文
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[8]崔尚金.猪瘟流行现状的分析与思考[J].畜禽业,2006,195:6-9.
[9]朱小甫,李晓成,陈德坤,等.猪瘟诊断技术研究进展[J].中国动物检疫,2007,24(2):45-47.
[10]胡慧,邱昌庆.猪瘟诊断和防制研究进展[J].中国兽医科技,2004,34(6):33-39.
[11]杨林,赵德明,熊永忠.猪瘟诊断技术的研究进展[J].畜牧兽医科技信息,2005,3:54-55.
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[14] Glodrick MA,Lowings JP,Ibala G.A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe(taqman)[J].J Virol Methods,1998,72(2):125-135.
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[17] Wensvoort G.Lelystad virus and the porcine epidemic abortion and respiratory syndrome[J].Vet Res,1993,24:117-124.
[18]刘光清,蔡雪晖,仇华吉,等.猪繁殖与呼吸综合征的研究进展[J].中国预防兽医学报,2001,23(1):72-76.
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[20]崔保安,刘占通,文英会.猪繁殖与呼吸综合征病毒分子生物学研究进展[J].动物医学进展,2004,25(1):22-24.
[21] Allende R,Lewis TL,Lu Z,et al.North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions[J].J Gen Virol,1999,80:370-315.
[22] Nelsen CJ,Murtaugh MP,Faaberg KS.Porcine reproductive and respiratory syndrome virus comparison:Divergent Evolution on Two Continents[J].J Virol,1999,73(1):270-280.
[23] Meulenberg JJ,Hulst MM,de Meijer EJ,et al.Lelystad virus,the causative agent of porcine epidemic abortion and respiratory syndrome(PEARS),is related to LDV and EAV[J].Virology,1993,192(1):62-72.
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[25] Plageman PG,Moennig V.Lactate dehydrogenase-elevating virus,equine arteritis virus and simian hemorrhagic fever virus,a new group of positive-strand RNA viruses[J].Adv virus Res,1992,41:99-192.
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