基于不同PCR方法的Ⅱ型鲤疱疹病毒检测技术研究
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摘要
异育银鲫"鳃出血病"是一种因感染了鲤疱疹病毒Ⅱ型(Cy HV-2)而引起的疾病,近年来给江苏异育银鲫养殖业造成了巨大的经济损失。为了能够建立及时、有效检出Ⅱ型鲤疱疹病毒的技术,本研究在克隆了Cy HV-2解旋酶基因和三联体蛋白基因的基础上,建立了检测CyHV-2的普通PCR、双重PCR、巢式PCR、环介导等温扩增、实时荧光定量PCR等方法,并对这5种PCR技术检测Cy HV-2的灵敏性进行了系统研究。结果表明,针对CyHV-2病毒的解旋酶基因和三联体蛋白基因,普通PCR能够检测出的极限值是2.4×10~4 copies/μL,双重PCR是1.4×10~4 copies/μL,巢式PCR是2.4×10~(-2)copies/μL,荧光定量PCR是2.4×10~(-2)copies/μL,环介导等温扩增是3.5×10~2 copies/μL。通过采用以上方法对从不同地区采集的54尾异育银鲫提取的DNA为模板进行临床检验,测定不同检测方法的阳性检出率。结果表明,实时荧光定量PCR和巢式PCR的检出率很高,分别为89%和90.7%;普通PCR的检出率最低,为68.5%。综合检测灵敏度和阳性检出率,环介导等温扩增(LAMP)是一种适用于生产实践的能有效检测CyHV-2的良好技术。
In recent years, gill hemorrhagic disease in the Carassius auratus gibelio, which is caused by the infection of CyHV-Ⅱ, has caused enormous economic losses to the breeding industry of gibel carp in Jiangsu. In order to establish the techniques to detect the CyHV-Ⅱ timely and effectivly, we set up ordinary PCR, duplex PCR, nested PCR, Loop-mediated isothermal amplification and Real-time quantitative PCR methods for detection of Cy HV-2 on the basis of cloning CyHV-2 helicase gene and triplet protein gene, and systematically studied the sensitivity of these five PCR techniques to detect CyHV-2. The results showed that for helicase gene and triplet protein gene of the Cy HV-Ⅱ, the limit value detected by ordinary PCR was 2.4×10~4 copies/μL; duplex PCR was 1.4×10~4 copies/μL;nested PCR was 2.4×10~(-2) copies/μL; Real-time quantitative PCR was 2.4×10~(-2) copies/μL; LAMP was 3.5×10~2 copies/μL.By adopting the above methods, the DNA extracted from 54 Carassius auratus gibelio collected from different places was used as template for clinical examination to determinate positive detection rates of different detection methods.The results showed that the detection rates of Real-time quantitative PCR and the nested PCR were 89% and90.7% respectively, which were high. Meanwhile, the detection rate of the ordinary PCR was 68.5% which was the lowest. Taking detection sensitivity and positive detection rate into consideration, LAMP is a good technique applicable to the production practice of detecting CyHV-2 effectively.
In recent years, gill hemorrhagic disease in the Carassius auratus gibelio, which is caused by the infection of CyHV-Ⅱ, has caused enormous economic losses to the breeding industry of gibel carp in Jiangsu. In order to establish the techniques to detect the CyHV-Ⅱ timely and effectivly, we set up ordinary PCR, duplex PCR, nested PCR, Loop-mediated isothermal amplification and Real-time quantitative PCR methods for detection of Cy HV-2 on the basis of cloning CyHV-2 helicase gene and triplet protein gene, and systematically studied the sensitivity of these five PCR techniques to detect CyHV-2. The results showed that for helicase gene and triplet protein gene of the Cy HV-Ⅱ, the limit value detected by ordinary PCR was 2.4×10~4 copies/μL; duplex PCR was 1.4×10~4 copies/μL;nested PCR was 2.4×10~(-2) copies/μL; Real-time quantitative PCR was 2.4×10~(-2) copies/μL; LAMP was 3.5×10~2 copies/μL.By adopting the above methods, the DNA extracted from 54 Carassius auratus gibelio collected from different places was used as template for clinical examination to determinate positive detection rates of different detection methods.The results showed that the detection rates of Real-time quantitative PCR and the nested PCR were 89% and90.7% respectively, which were high. Meanwhile, the detection rate of the ordinary PCR was 68.5% which was the lowest. Taking detection sensitivity and positive detection rate into consideration, LAMP is a good technique applicable to the production practice of detecting CyHV-2 effectively.
引文
Goodwin A.E.,Merry G.E.,and Sadler J.,2006,Detection of the herpesviral hematopoietic necrosis disease agent(cyprinid herpesvirus 2)in moribund and healthy goldfish:validation of a quantitative PCR diagnostic method,Diseases of Aquatic Organisms,69(2-3):137-143
Goodwin A.E.,Sadler J.,Merry G.E.,and Marecaux E.N.,2009,Herpesviral haematopoietic necrosis virus(CyHV-2)infection:case studies from commercial goldfish farms,Journal of Fish Diseases,32(3):271-278
Guo Z.,Zhang D.,Ma H.,Su Y.,Feng J.,and Xu L.,2014,Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification,J.Virol.Methods,208:171-176
He J.,Shi X.,Yu L.,Zheng X.,Lan W.,Jia P.,Wang J.,and Liu H.,2013,Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2,J.Virol.Methods,194(1-2):206-210
Jeffery K.R.,Bateman K.,Bayley A.,Feist S.W.,Hulland J.,Longshaw C.,Stone D.,Woolford G.,and Way K.,2007,Isolation of a cyprinid herpesvirus 2 from goldfish,Carassius auratus(L.),in the UK,Journal of Fish Diseases,30(11):649-656
Jung S.J.,and Miyazaki T.,1995,Herpesviral haematopoietic necrosis of goldfish,Carassius auratus(L.),Journal of Fish Diseases,18(3):211-220
Liang L.G.,Xie J.,and Luo D.,2014,Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification,Letters in Applied Microbiology,59(4):432-437
Liu X.X.,Gen Y.,Zhou Z.Y.,Wang K.Y.,Huang X.L.,Cheng D.F.,and Zhou Y.,2014,Development of a nested PCR detection method for Chinese giant salamanders ranavirus,Zhongguo Shouyi Xuebao(Chinese Journal of Veterinary Science),34(3):406-410(刘星星,耿毅,周赵英,汪开毓,黄小丽,陈德芳,周燕,2014,大鲵蛙病毒巢氏PCR检测方法的建立,中国兽医学报,34(3):406-410)
Luo D.,2014,Development of four methods for detection of Cyprinod herpesvirus 2,Thesis for M.S.,College of Animal Science and Technology,Gansu Agricultural University,Supervisor:Wu X.D.,pp.11-15(罗丹,2014,鲤疱疹病毒Ⅱ型4种检测方法的建立及应用,硕士学位论文,甘肃农业大学动物科技学院,导师:吴旭东,pp.11-15)
Luo D.,Liang L.G.,Xie J.,and Wu X.D.,2014,Development of duplex PCR for detection of Cyprinid herpesvirus 2,Zhongguo Yufang Shouyi Xuebao(Chinese Journal of Preventive Veterinary Medicine),36(5):379-382(罗丹,梁利国,谢骏,吴旭东,2014,2型鲤疱疹病毒双重PCR快速检测方法的建立及应用,中国预防兽医学报,36(5):379-382)
Luo D.,Liang L.G.,Xie J.,and Wu X.D.,2014,Research progress of cyprinid herpesvirusⅠ,Ⅱ,Ⅲ,Shui Shengtaixue Zazhi(Journal of Hydroecology),2014(3):94-100(罗丹,梁利国,谢骏,吴旭东,2014,鲤疱疹病毒Ⅰ,Ⅱ,Ⅲ型研究进展,水生态学杂志,2014(3):94-100)
Ren W.G.,Wang C.M.,and Li Y.,2007,Real-time fluorescent quantitative PCR technique and its application in aquaculture,Haiyang Shuichan Yanjiu(Marine Fisheries Research),28(6):97-103(任伟成,王崇明,李赟,2007,荧光实时定量PCR技术及其在水产养殖研究中的应用,海洋水产研究,28(6):97-103)
Soliman H.,and El-Matbouli M.,2009,Immunocapture and direct bnding loop mediated isothermal amplification simplify molecular diagnosis of cyprinid herpesvirus-3,J.Virol.Methods,162(1-2):91-95
Stephens F.J,Raidal S.R.,and Jones B.,2004,Haematopoietic necrosis in a goldfish(Carassius auratus)associated with an agent morphologically similar to herpesvirus,Australian Veterinary Journal,82(3):167-169
Wang L.,He J.,Liang L.,Zheng X.,Jia P.,Shi X.,Lan W.,Xie J.,Liu H.,and Xu P.,2012,Mass mortality caused by cyprinid herpesvirus 2(Cy HV-Ⅱ)in prussian carp(Carassius gibelio)in China,Fish Pathology,32(5):164-173
Wei L.L.,2012,Real-time PCR technology and its application in fish diseases,Shengwujishu(Biotechnology),22(5):82-85(隗黎丽,2012,实时荧光定量PCR技术在鱼类病害研究中的应用,生物技术,22(5):82-85)
Whelan J.A.,Russeu N.B.,and Whelan M.A.,2003,A method for the absolute quattification of cDNA using real-time PCR,Journal of Immunological Methods,278:261-269
Xiao G.X.,Li Z.J.,Meng X.H.,Zhang Q.W.,Luo K.,and Kong J.,2011,Sensitivity comparison of three PCR-based methods for white spot syndrome virus(WSSV)detection,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),18(3):667-673(肖广侠,李战军,孟宪红,张庆文,罗坤,孔杰,2011,白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较,中国水产科学,18(3):667-673)
Yue Z.Q.,Liu H.,Liang C.Z.,Gao H.W.,Xu B.,Deng M.J.,and Jiang Y.L.,2008,Real-time quantitative RT-PCR assay for detection of IHNV in fish,Shuisheng Shengwu Xuebao(Acta Hydrobiologica Sinica),32(1):91-95(岳志芹,刘荭,梁成珠,高宏伟,徐彪,邓明俊,江育林,2008,实时定量RT-PCR检测鱼类传染性造血器官坏死病毒方法的建立与应用,水生生物学报,32(1):91-95)
Zhang L.,Yin Y.P.,and Wang Z.K.,2009,The advances and development of loop-mediated isothermal amplification method,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),37(6):21-25(张仑,殷幼平,王中康,2009,环介导等温扩增技术的研究进展,贵州农业科学,37(6):21-25)
Zhang Z.G.,Xu L.P.,Wang S.,Wang J.B.,Wang X.L.,and Cao H.,2011,Detection of koi herpesvirus by duplex PCR assay,Zhongguo Xumu Shouyi(Chinese Animal Husbandry and Veterinary Medicine),38(9):83-86(张振国,徐立蒲,王姝,王静波,王小亮,曹欢,2011,锦鲤疱疹病毒双重PCR检测方法的研究,中国畜牧兽医,38(9):83-86)
Zhao Z.,Ren C.,Jiang X.,Zhang L.,Fen J.,and Hu C.,2010,Comparative study of specificity and sensitivity for IHHNVdetection between real-time PCR and LAMP methods,Shuisheng Shengwu Xuebao(Acta Hydrobiologica Sinica),34(5):984-989(赵哲,任春华,江晓,张吕平,冯娟,胡超群,2010,荧光定量PCR与LAMP检测IHHNV的特异性和灵敏性比较,水生生物学报,34(5):984-989)
Z hong C.Y.,and Sun M.,2015,Determination of the copynumber of the resident plasmid pBMB67 from Bacillus thuringiensis strain YBT-1520 by quanti-tative Real-time PCR,Hubei Nongye Kexue(Hubei Agricultural Science),(8):2001-2006(钟春英,孙明,2015,实时定量PCR测定苏云金芽胞杆菌质粒pBMB67的拷贝数,湖北农业科学,(8):2001-2006)
Goodwin A.E.,Sadler J.,Merry G.E.,and Marecaux E.N.,2009,Herpesviral haematopoietic necrosis virus(CyHV-2)infection:case studies from commercial goldfish farms,Journal of Fish Diseases,32(3):271-278
Guo Z.,Zhang D.,Ma H.,Su Y.,Feng J.,and Xu L.,2014,Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification,J.Virol.Methods,208:171-176
He J.,Shi X.,Yu L.,Zheng X.,Lan W.,Jia P.,Wang J.,and Liu H.,2013,Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2,J.Virol.Methods,194(1-2):206-210
Jeffery K.R.,Bateman K.,Bayley A.,Feist S.W.,Hulland J.,Longshaw C.,Stone D.,Woolford G.,and Way K.,2007,Isolation of a cyprinid herpesvirus 2 from goldfish,Carassius auratus(L.),in the UK,Journal of Fish Diseases,30(11):649-656
Jung S.J.,and Miyazaki T.,1995,Herpesviral haematopoietic necrosis of goldfish,Carassius auratus(L.),Journal of Fish Diseases,18(3):211-220
Liang L.G.,Xie J.,and Luo D.,2014,Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification,Letters in Applied Microbiology,59(4):432-437
Liu X.X.,Gen Y.,Zhou Z.Y.,Wang K.Y.,Huang X.L.,Cheng D.F.,and Zhou Y.,2014,Development of a nested PCR detection method for Chinese giant salamanders ranavirus,Zhongguo Shouyi Xuebao(Chinese Journal of Veterinary Science),34(3):406-410(刘星星,耿毅,周赵英,汪开毓,黄小丽,陈德芳,周燕,2014,大鲵蛙病毒巢氏PCR检测方法的建立,中国兽医学报,34(3):406-410)
Luo D.,2014,Development of four methods for detection of Cyprinod herpesvirus 2,Thesis for M.S.,College of Animal Science and Technology,Gansu Agricultural University,Supervisor:Wu X.D.,pp.11-15(罗丹,2014,鲤疱疹病毒Ⅱ型4种检测方法的建立及应用,硕士学位论文,甘肃农业大学动物科技学院,导师:吴旭东,pp.11-15)
Luo D.,Liang L.G.,Xie J.,and Wu X.D.,2014,Development of duplex PCR for detection of Cyprinid herpesvirus 2,Zhongguo Yufang Shouyi Xuebao(Chinese Journal of Preventive Veterinary Medicine),36(5):379-382(罗丹,梁利国,谢骏,吴旭东,2014,2型鲤疱疹病毒双重PCR快速检测方法的建立及应用,中国预防兽医学报,36(5):379-382)
Luo D.,Liang L.G.,Xie J.,and Wu X.D.,2014,Research progress of cyprinid herpesvirusⅠ,Ⅱ,Ⅲ,Shui Shengtaixue Zazhi(Journal of Hydroecology),2014(3):94-100(罗丹,梁利国,谢骏,吴旭东,2014,鲤疱疹病毒Ⅰ,Ⅱ,Ⅲ型研究进展,水生态学杂志,2014(3):94-100)
Ren W.G.,Wang C.M.,and Li Y.,2007,Real-time fluorescent quantitative PCR technique and its application in aquaculture,Haiyang Shuichan Yanjiu(Marine Fisheries Research),28(6):97-103(任伟成,王崇明,李赟,2007,荧光实时定量PCR技术及其在水产养殖研究中的应用,海洋水产研究,28(6):97-103)
Soliman H.,and El-Matbouli M.,2009,Immunocapture and direct bnding loop mediated isothermal amplification simplify molecular diagnosis of cyprinid herpesvirus-3,J.Virol.Methods,162(1-2):91-95
Stephens F.J,Raidal S.R.,and Jones B.,2004,Haematopoietic necrosis in a goldfish(Carassius auratus)associated with an agent morphologically similar to herpesvirus,Australian Veterinary Journal,82(3):167-169
Wang L.,He J.,Liang L.,Zheng X.,Jia P.,Shi X.,Lan W.,Xie J.,Liu H.,and Xu P.,2012,Mass mortality caused by cyprinid herpesvirus 2(Cy HV-Ⅱ)in prussian carp(Carassius gibelio)in China,Fish Pathology,32(5):164-173
Wei L.L.,2012,Real-time PCR technology and its application in fish diseases,Shengwujishu(Biotechnology),22(5):82-85(隗黎丽,2012,实时荧光定量PCR技术在鱼类病害研究中的应用,生物技术,22(5):82-85)
Whelan J.A.,Russeu N.B.,and Whelan M.A.,2003,A method for the absolute quattification of cDNA using real-time PCR,Journal of Immunological Methods,278:261-269
Xiao G.X.,Li Z.J.,Meng X.H.,Zhang Q.W.,Luo K.,and Kong J.,2011,Sensitivity comparison of three PCR-based methods for white spot syndrome virus(WSSV)detection,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),18(3):667-673(肖广侠,李战军,孟宪红,张庆文,罗坤,孔杰,2011,白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较,中国水产科学,18(3):667-673)
Yue Z.Q.,Liu H.,Liang C.Z.,Gao H.W.,Xu B.,Deng M.J.,and Jiang Y.L.,2008,Real-time quantitative RT-PCR assay for detection of IHNV in fish,Shuisheng Shengwu Xuebao(Acta Hydrobiologica Sinica),32(1):91-95(岳志芹,刘荭,梁成珠,高宏伟,徐彪,邓明俊,江育林,2008,实时定量RT-PCR检测鱼类传染性造血器官坏死病毒方法的建立与应用,水生生物学报,32(1):91-95)
Zhang L.,Yin Y.P.,and Wang Z.K.,2009,The advances and development of loop-mediated isothermal amplification method,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),37(6):21-25(张仑,殷幼平,王中康,2009,环介导等温扩增技术的研究进展,贵州农业科学,37(6):21-25)
Zhang Z.G.,Xu L.P.,Wang S.,Wang J.B.,Wang X.L.,and Cao H.,2011,Detection of koi herpesvirus by duplex PCR assay,Zhongguo Xumu Shouyi(Chinese Animal Husbandry and Veterinary Medicine),38(9):83-86(张振国,徐立蒲,王姝,王静波,王小亮,曹欢,2011,锦鲤疱疹病毒双重PCR检测方法的研究,中国畜牧兽医,38(9):83-86)
Zhao Z.,Ren C.,Jiang X.,Zhang L.,Fen J.,and Hu C.,2010,Comparative study of specificity and sensitivity for IHHNVdetection between real-time PCR and LAMP methods,Shuisheng Shengwu Xuebao(Acta Hydrobiologica Sinica),34(5):984-989(赵哲,任春华,江晓,张吕平,冯娟,胡超群,2010,荧光定量PCR与LAMP检测IHHNV的特异性和灵敏性比较,水生生物学报,34(5):984-989)
Z hong C.Y.,and Sun M.,2015,Determination of the copynumber of the resident plasmid pBMB67 from Bacillus thuringiensis strain YBT-1520 by quanti-tative Real-time PCR,Hubei Nongye Kexue(Hubei Agricultural Science),(8):2001-2006(钟春英,孙明,2015,实时定量PCR测定苏云金芽胞杆菌质粒pBMB67的拷贝数,湖北农业科学,(8):2001-2006)
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