PCR结合核酸斑点杂交检测猪伪狂犬病毒
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摘要
本研究基于猪伪狂犬病毒(Porcine pseudorabies virus,PRV)gE基因,建立了一种PCR结合核酸斑点杂交检测方法来鉴别野毒株和疫苗株,并利用这一方法对山东省部分地区的猪临床病料中PRV进行了检测。结果显示,建立的PCR结合核酸斑点杂交检测方法只与PRV野毒株反应,而与PRV gE基因缺失疫苗、猪瘟病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒以及猪圆环病毒2型不反应。PCR结合核酸斑点杂交方法在53份病料中检出PRV阳性病料5份,阳性率为9.43%,高于单纯PCR的检出率(7.55%)。结果表明,本研究建立的PCR结合斑点杂交的方法特异性强,灵敏度高,适合PRV野毒的检测以及流行病学调查,可应用于PRV的鉴别诊断和净化。
In the present study, a combined PCR method with nucleic acid dot blot hybridization was developed for differentiation of Pseudorabies virus(PRV) vaccine strains from wild viruses based on the conserved sequences of gE gene. The results showed that the the combined PCR method with nucleic acid dot blot hybridization reacted with PRV wild-type viruses but not with gE gene deleted vaccine strains, Classical swine fever virus(CSFV), Swine epidemic diarrhea virus(PEDV), Porcine reproductive and respiratory syndrome virus(PRRSV) and porcine circovirus type 2(PCV2). Afterwards, 53 samples were collected from diseased pigs in different regions of Shandong province for PRV diagnosis. As a result, 9.43%(5/53) of these samples were PRV positive by the combined PCR with nucleic acid dot blot hybridization, while 7.55%(4/53) samples were positive by PCR only. These results suggest that this combined PCR method with nucleic acid dot blot hybridization might be used for differential diagnosis and epidemiological investigation of PRV.
In the present study, a combined PCR method with nucleic acid dot blot hybridization was developed for differentiation of Pseudorabies virus(PRV) vaccine strains from wild viruses based on the conserved sequences of gE gene. The results showed that the the combined PCR method with nucleic acid dot blot hybridization reacted with PRV wild-type viruses but not with gE gene deleted vaccine strains, Classical swine fever virus(CSFV), Swine epidemic diarrhea virus(PEDV), Porcine reproductive and respiratory syndrome virus(PRRSV) and porcine circovirus type 2(PCV2). Afterwards, 53 samples were collected from diseased pigs in different regions of Shandong province for PRV diagnosis. As a result, 9.43%(5/53) of these samples were PRV positive by the combined PCR with nucleic acid dot blot hybridization, while 7.55%(4/53) samples were positive by PCR only. These results suggest that this combined PCR method with nucleic acid dot blot hybridization might be used for differential diagnosis and epidemiological investigation of PRV.
引文
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[3]Pomeranz L E,Reynolds A E,Hengartner C J.Molecular biology of pseudorabies virus:impact on neurovirology and veterinary medicine[J].Microbiol Mol Biol Rev,2005,69(3):462-500.
[4]程晶,陈艳红,荫硕焱,等.2006~2008年我国部分地区规模化猪场PRV血清流行病学调查[J].中国动物传染病学报,2009,17(1):67-72.
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[7]Ferrari M,Brack A,Romanelli M G,et al.A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus(PRV)mutant inoculated by different routes to protect pigs against PRV infection[J].J Vet Med B Infect Dis Vet Public Health,2000,47(10):753-762.
[8]刘正飞,陈焕春,吴斌,等.伪狂犬病病毒鄂A株TK-/gE-/gI-基因缺失疫苗的安全性和保护力研究[J].畜牧兽医学报,2004,35(1):70-73.
[9]武吉强,徐晶晶,童武,等.伪狂犬病病毒变异株gE蛋白单克隆抗体的制备与鉴定[J].中国动物传染病学报,2017,25(3):23-27.
[10]Wang Y,Qiao S,Li X,et al.Molecular epidemiology of outbreak-associated pseudorabies virus(PRV)strains in central China[J].Virus Genes,2015,50(3):401-409.
[11]Chiari M,Ferrari N,Bertoletti M,et al.Long-term surveillance of Aujeszky’s disease in the alpine wild boar(Sus scrofa)[J].Ecohealth,2015,12(4):563-570.
[12]Grau-Roma L,Fraile L,Segalés J.Recent advances in the epidemiology,diagnosis and control of diseases caused by porcine circovirus type 2[J].Vet J,2011,187(1):23-32.
[13]金升藻,陈焕春,熊符.伪狂犬病基因缺失疫苗研究进展[J].中国农业科学,2002,35(1):89-93.
[14]黄元,陈晶,赵翠玲,等.伪狂犬病病毒变异株的鉴定及伪狂犬病的防控[J].中国畜牧兽医,2016,43(9):2461-2467.
[15]Guo G J,Lu S F,Qu G G,et al.Expression of pseudorabies virus gE core epitopes in Escherichia coli strain BL21 and utilization of indirect PRV gE-ELISA[J].Agr Biotechnol,2014,3(4):39-44.
[16]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报,2015,23(3):1-6.
[17]杨睿,杨金龙,王孝友,等.快速诊断伪狂犬病毒的LAMP方法的建立[J].中国兽医学报,2011,31(2):161-164.
[18]唐勇,陈焕春,覃雅丽,等.伪狂犬病毒gG基因的表达及gG-LAT诊断方法的建立[J].畜牧兽医学报,2003,34(1):72-76.
[19]崔治中,赵鹏,孙淑红,等.鸡致病性外源性禽白血病病毒特异性核酸探针交叉斑点杂交检测试剂盒的研制[J].中国兽药杂志,2011,45(8):5-11.
[2]Mettenleiter T C.Aujeszky's disease(pseudorabies)virus:the virus and molecular pathogenesis-state of the art[J].Vet Res,2000,31(1):99-115.
[3]Pomeranz L E,Reynolds A E,Hengartner C J.Molecular biology of pseudorabies virus:impact on neurovirology and veterinary medicine[J].Microbiol Mol Biol Rev,2005,69(3):462-500.
[4]程晶,陈艳红,荫硕焱,等.2006~2008年我国部分地区规模化猪场PRV血清流行病学调查[J].中国动物传染病学报,2009,17(1):67-72.
[5]王林青,郑兰兰,李坤,等.猪伪狂犬病病毒载体重组疫苗研究进展[J].中国预防兽医学报,2014,36(2):160-164.
[6]王寅彪.猪伪狂犬病野毒感染与疫苗免疫鉴别诊断方法的建立及初步临床应用[D].长春:吉林大学,2015.
[7]Ferrari M,Brack A,Romanelli M G,et al.A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus(PRV)mutant inoculated by different routes to protect pigs against PRV infection[J].J Vet Med B Infect Dis Vet Public Health,2000,47(10):753-762.
[8]刘正飞,陈焕春,吴斌,等.伪狂犬病病毒鄂A株TK-/gE-/gI-基因缺失疫苗的安全性和保护力研究[J].畜牧兽医学报,2004,35(1):70-73.
[9]武吉强,徐晶晶,童武,等.伪狂犬病病毒变异株gE蛋白单克隆抗体的制备与鉴定[J].中国动物传染病学报,2017,25(3):23-27.
[10]Wang Y,Qiao S,Li X,et al.Molecular epidemiology of outbreak-associated pseudorabies virus(PRV)strains in central China[J].Virus Genes,2015,50(3):401-409.
[11]Chiari M,Ferrari N,Bertoletti M,et al.Long-term surveillance of Aujeszky’s disease in the alpine wild boar(Sus scrofa)[J].Ecohealth,2015,12(4):563-570.
[12]Grau-Roma L,Fraile L,Segalés J.Recent advances in the epidemiology,diagnosis and control of diseases caused by porcine circovirus type 2[J].Vet J,2011,187(1):23-32.
[13]金升藻,陈焕春,熊符.伪狂犬病基因缺失疫苗研究进展[J].中国农业科学,2002,35(1):89-93.
[14]黄元,陈晶,赵翠玲,等.伪狂犬病病毒变异株的鉴定及伪狂犬病的防控[J].中国畜牧兽医,2016,43(9):2461-2467.
[15]Guo G J,Lu S F,Qu G G,et al.Expression of pseudorabies virus gE core epitopes in Escherichia coli strain BL21 and utilization of indirect PRV gE-ELISA[J].Agr Biotechnol,2014,3(4):39-44.
[16]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报,2015,23(3):1-6.
[17]杨睿,杨金龙,王孝友,等.快速诊断伪狂犬病毒的LAMP方法的建立[J].中国兽医学报,2011,31(2):161-164.
[18]唐勇,陈焕春,覃雅丽,等.伪狂犬病毒gG基因的表达及gG-LAT诊断方法的建立[J].畜牧兽医学报,2003,34(1):72-76.
[19]崔治中,赵鹏,孙淑红,等.鸡致病性外源性禽白血病病毒特异性核酸探针交叉斑点杂交检测试剂盒的研制[J].中国兽药杂志,2011,45(8):5-11.