检测鹿黏膜病病毒LDR-PCR方法的建立
详细信息 查看官网全文
- 英文论文题名：Development of LDR-PCR assay for deer mucosal disease
- 论文作者：王雪 ; 刘新鑫 ; 穆昱 ; 尹仁福 ; 丁壮
- 英文论文作者：Wang Xue ; Liu Xinxin ; Mu Yu ; Yin Renfu ; Ding Zhuang ; College of Veterinary Medicine ; Jilin University
- 年：2016
- 作者机构：吉林大学动物医学学院;
- 论文关键词：LDR-PCR ; 鹿黏膜病病毒 ; 条件优化 ; 检测
- 英文论文关键词：LDR-PCR ; deer mucosal disease virus ; optimization ; detection
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S858.25
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：9
- 文件大小：920k
- 原文格式：O
摘要
为准确特异灵敏地检测鹿黏膜病病毒(BVDV),本研究建立了一种新的LDR-PCR方法。首先在病毒的保守区内设计1对LDR探针,LDR探针两端各连有1段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,本方法可以特异地检测BVDV,最低检测限为10~1个拷贝。此方法的建立为BVDV基础研究和临床应用提供了良好的技术平台,为进一步开发高通量的多病原检测技术提供了技术基础。
In order to accurately specific and sensitive detection of deer mucosal disease virus(BVDV),this study established a new LDR-PCR method.First,the conservative region of the virus to design a pair LDR probes,LDR probes attached at each end of the corresponding period of the primer sequences to the ligation product as a template PCR,agarose gel electrophoresis test results.By LDR reaction annealing temperature,the ligase concentration and reaction conditions such as probe concentrations were optimized to determine the optimum LDR reaction system,and the establishment of LDR-PCR method.The results show that this approach can specifically detect BVDV,the lowest detection limit of 10 copies.This method is an BVDV establish basic research and clinical applications provide a good technical platform,provide the technical foundation for the further development of high-throughput multi-pathogen detection technology.
In order to accurately specific and sensitive detection of deer mucosal disease virus(BVDV),this study established a new LDR-PCR method.First,the conservative region of the virus to design a pair LDR probes,LDR probes attached at each end of the corresponding period of the primer sequences to the ligation product as a template PCR,agarose gel electrophoresis test results.By LDR reaction annealing temperature,the ligase concentration and reaction conditions such as probe concentrations were optimized to determine the optimum LDR reaction system,and the establishment of LDR-PCR method.The results show that this approach can specifically detect BVDV,the lowest detection limit of 10 copies.This method is an BVDV establish basic research and clinical applications provide a good technical platform,provide the technical foundation for the further development of high-throughput multi-pathogen detection technology.
引文
[1]Qi T and Cui SJ.Expression,purification,and characterization of recombinant NS-1,the porcine parvovirus non-structural protein[J].J Virol Methods,2009,157(1):93-97.
[2]Sharma R and Saikumar G.Porcine parvovirus-and porcine circovirus 2-associated reproductive failure and neonatal mortality in crossbred Indian pigs[J].Trop Anim Health Prod,2009,42(3):515-522.
[3]Wolf V H,Menossi M,Mourao G B,et al.Molecular basis for porcine parvovirus detection in dead fetuses[J].Genet Mol Res,2008,7(2):509-517.
[4]Chen CM and Cui SJ.Detection of porcine parvovirus by loop-mediated isothermal amplification.J Virol Methods,2009,155(2):122-125.
[5]Cai JP,Wang YD,Tse H,et al.Detection of asymptomatic antigenemia in pigs infected by porcine reproductive and respiratory syndrome virus(PRRSV)by a novel capture immunoassay with monoclonal antibodies against the nucleocapsid protein of PRRSV[J].Clin Vaccine Immunol,2009,16(12):1 822-1 828.
[6]Klinge KL,Vaughn EM,Roof MB,et al.Age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine[J].Virol J,2009,6(1):177.
[7]Rossow KD.Porcine reproductive and respiratory syndrome[J].Vet Pathol,1998,35(1):1-20.
[8]Wootton S,Yoo D and Rogan D.Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus(PRRSV)isolate[J].Arch Virol,2000,145(11):2 297-2 323.
[9]Girigoswami A,Jung C,Mun HY,et al.PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction[J].J Biochem Biophys Methods,2008,70(6):897-902.
[10]Yi P,Chen Z,Zhao Y,et al.PCR/LDR/capillary electrophoresis for detection of single-nucleotide differences between fetal and maternal DNA in maternal plasma[J].Prenat Diagn,2009,29(3):217-222.
[11]Niederhauser C,Kaempf L and Heinzer I.Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases[J].Eur J Clin Microbiol Infect Dis,2000,19(6):477-480.
[12]Das S,Pingle MR,Munoz-Jordan J,et al.Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay[J].J Clin Microbiol,2008,46(10):3 276-3 284.
[13]张世扬,肖振贤,赵建龙,等.基于连接酶检测反应的并行分型系统检测agt M235t和ace I/D基因多态性.华东理工大学学报(自然科学版),2006,32(9):1 050-1 054.
[14]李灵敏,曹志中,沈霖德.基于连接酶检测反应的cgrp和il-1a基因多态与重度慢性牙周炎的相关性研究.牙体牙髓牙周病学杂志,2008,18(5):241-246.
[15]王刚,李凯,周宇苟,等.新型通用探针LDR分型技术的开发及细胞色素p450基因多位点分型.华东理工大学学报(自然科学版),2008,34(4):503-508.
[16]Rondini S,Pingle MR,Das S,et al.Development of multiplex PCR-ligase detection reaction assay for detection of west nile virus[J].J Clin Microbiol,2008,46(7):2 269-2 279.
[17]刘业冰,张磊,宁宜宝,等.猪细小病毒LAMP检测方法的建立[J].中国农业科学,2010,43(14):3 012-3 018.
[18]李明凤,魏战勇,王学斌,等.猪细小病毒SYBR Green I实时定量PCR检测方法的建立[J].浙江农业学报,2009,21(3):220-224.
[19]俞琼.应用LDR技术探讨cPLA2家族基因多态性与精神分裂症的关系[D].长春:吉林大学.2007.
[2]Sharma R and Saikumar G.Porcine parvovirus-and porcine circovirus 2-associated reproductive failure and neonatal mortality in crossbred Indian pigs[J].Trop Anim Health Prod,2009,42(3):515-522.
[3]Wolf V H,Menossi M,Mourao G B,et al.Molecular basis for porcine parvovirus detection in dead fetuses[J].Genet Mol Res,2008,7(2):509-517.
[4]Chen CM and Cui SJ.Detection of porcine parvovirus by loop-mediated isothermal amplification.J Virol Methods,2009,155(2):122-125.
[5]Cai JP,Wang YD,Tse H,et al.Detection of asymptomatic antigenemia in pigs infected by porcine reproductive and respiratory syndrome virus(PRRSV)by a novel capture immunoassay with monoclonal antibodies against the nucleocapsid protein of PRRSV[J].Clin Vaccine Immunol,2009,16(12):1 822-1 828.
[6]Klinge KL,Vaughn EM,Roof MB,et al.Age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine[J].Virol J,2009,6(1):177.
[7]Rossow KD.Porcine reproductive and respiratory syndrome[J].Vet Pathol,1998,35(1):1-20.
[8]Wootton S,Yoo D and Rogan D.Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus(PRRSV)isolate[J].Arch Virol,2000,145(11):2 297-2 323.
[9]Girigoswami A,Jung C,Mun HY,et al.PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction[J].J Biochem Biophys Methods,2008,70(6):897-902.
[10]Yi P,Chen Z,Zhao Y,et al.PCR/LDR/capillary electrophoresis for detection of single-nucleotide differences between fetal and maternal DNA in maternal plasma[J].Prenat Diagn,2009,29(3):217-222.
[11]Niederhauser C,Kaempf L and Heinzer I.Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases[J].Eur J Clin Microbiol Infect Dis,2000,19(6):477-480.
[12]Das S,Pingle MR,Munoz-Jordan J,et al.Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay[J].J Clin Microbiol,2008,46(10):3 276-3 284.
[13]张世扬,肖振贤,赵建龙,等.基于连接酶检测反应的并行分型系统检测agt M235t和ace I/D基因多态性.华东理工大学学报(自然科学版),2006,32(9):1 050-1 054.
[14]李灵敏,曹志中,沈霖德.基于连接酶检测反应的cgrp和il-1a基因多态与重度慢性牙周炎的相关性研究.牙体牙髓牙周病学杂志,2008,18(5):241-246.
[15]王刚,李凯,周宇苟,等.新型通用探针LDR分型技术的开发及细胞色素p450基因多位点分型.华东理工大学学报(自然科学版),2008,34(4):503-508.
[16]Rondini S,Pingle MR,Das S,et al.Development of multiplex PCR-ligase detection reaction assay for detection of west nile virus[J].J Clin Microbiol,2008,46(7):2 269-2 279.
[17]刘业冰,张磊,宁宜宝,等.猪细小病毒LAMP检测方法的建立[J].中国农业科学,2010,43(14):3 012-3 018.
[18]李明凤,魏战勇,王学斌,等.猪细小病毒SYBR Green I实时定量PCR检测方法的建立[J].浙江农业学报,2009,21(3):220-224.
[19]俞琼.应用LDR技术探讨cPLA2家族基因多态性与精神分裂症的关系[D].长春:吉林大学.2007.