强、弱毒力新城疫病毒双重荧光定量RT-PCR检测方法的建立
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摘要
通过比对新城疫病毒强、弱毒株F基因序列,根据其在F0裂解位点的差异以及F基因保守序列设计合成NDV-V-probe和NDV-L-probe探针组和NDV-F、NDV-R引物组,以基因Ⅶ型NA-1株和基因Ⅱ型LaSota疫苗株病毒RNA作为定量检测的标准品,建立检测方法。对所建立的标准曲线进行分析,相关系数R2均为0.997,线性关系良好。通过计算变异系数CV/%分别为0.034%和0.027%,均小于1%,说明稳定性良好。对禽类常见病毒IBV、H9亚型AIV检测未发现有交叉反应,特异性好、重复性佳。结果表明:成功建立了新城疫病毒强、弱毒双重荧光定量RT-PCR的检测方法,实现了从分子水平上快速鉴别强、弱毒力的新城疫病毒,也可快速区分野毒感染动物和疫苗接种动物,减少不必要的经济损失。
In this study,we compared velogenic NDV F gene sequence with that of lentogenic,and then designed probes and primers according to the differences in the F0 cleavage site and the conserved sequences of F gene.NDV-F & NDV-R were selected as the best primer sets and NDV-Vprobe & NDV-L-probe were selected as the best probe sets after screening groups of probes and primers.the genotypeⅦ Newcastle disease virus NA-1strain and the vaccine strain LaSota virus RNA were respectively treated as standard for the development of a duplex fluorescence quantitative RT-PCR detection method.The correlation coefficient R2 of the established standard was0.997,with a good linear relationship,and the coefficient of variation(CV/%)were 0.034% and0.027%respectively,which were less than 1%,indicating agood stability.Avian virus IBV,H9-AIV were not found cross-reactivity,indicating its good specificity and repeatability.Taken together,NDV velogenic and lentogenic strains can be distinguished rapidly using a duplex fluorescence quantitative RT-PCR detection system,meanwhile,the detection system allows a strategy of differentiating infected from vaccinated animals,which could reduce unnecessary economic losses.
In this study,we compared velogenic NDV F gene sequence with that of lentogenic,and then designed probes and primers according to the differences in the F0 cleavage site and the conserved sequences of F gene.NDV-F & NDV-R were selected as the best primer sets and NDV-Vprobe & NDV-L-probe were selected as the best probe sets after screening groups of probes and primers.the genotypeⅦ Newcastle disease virus NA-1strain and the vaccine strain LaSota virus RNA were respectively treated as standard for the development of a duplex fluorescence quantitative RT-PCR detection method.The correlation coefficient R2 of the established standard was0.997,with a good linear relationship,and the coefficient of variation(CV/%)were 0.034% and0.027%respectively,which were less than 1%,indicating agood stability.Avian virus IBV,H9-AIV were not found cross-reactivity,indicating its good specificity and repeatability.Taken together,NDV velogenic and lentogenic strains can be distinguished rapidly using a duplex fluorescence quantitative RT-PCR detection system,meanwhile,the detection system allows a strategy of differentiating infected from vaccinated animals,which could reduce unnecessary economic losses.
引文
[1]中华人民共和国国务院.[2012]31号.国务院办公厅关于印发国家中长期动物疫病防治规划(2012-2020年)的通知[G].北京:中华人民共和国国务院,2012.
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[3]尹仁福,刘新鑫,丁壮,等.鹅源新城疫病毒实时荧光定量PCR检测方法的建立[J].中国兽医科学,2008,38(4):320-322.
[4]明文龙,尹仁福,谢光耀,等.鉴别新城疫强,弱毒一步RT-PCR方法的建立及应用[J].中国兽医学报,2015,35(7):1056-1059.
[5]DIMITROV K M,RAMEY A M,QIU X,et al.Temporal,geographic,and host distribution of avian paramyxovirus 1(Newcastle disease virus)[J].Infect Gene Evol,2016,39:22-34.
[6]NATH B,BARMAN N N,KUMAR S.Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-2015[J].Microbial Pathog,2016,91:85-91.
[7]SAMADI S,KIANIZADEH M,NAJAFI M F,et al.Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran[J].Virus Gene,2014,48(2):290-295.
[8]丁壮,王承宇,向华,等.鹅副黏病毒分离株生物学特性的研究[J].中国预防兽医学报,2002,24(5):390-391.
[9]ALEXANDER D.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Tech,2000,19(2):443-455.
[10]丁壮,尹仁福,薛聪,等.新城疫流行病学新特点及鹅新城疫防控策略[J].中国兽医学报,2015,35(1):159-168.
[11]HU S,MA H,WU Y,et al.A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics[J].Vaccine,2009,27(6):904-910.
[12]CHONG Y L,PADHI A,HUDSON P J,et al.The effect of vaccination on the evolution and population dynamics of avian paramyxovirus-1[J].PLoS Pathog,2010,6(4):e1000872.
[2]曹殿军,刘培欣.鸡新城疫病毒强弱毒株RT-PCR鉴别诊断方法[J].中国预防兽医学报,2000,22(6):415-417.
[3]尹仁福,刘新鑫,丁壮,等.鹅源新城疫病毒实时荧光定量PCR检测方法的建立[J].中国兽医科学,2008,38(4):320-322.
[4]明文龙,尹仁福,谢光耀,等.鉴别新城疫强,弱毒一步RT-PCR方法的建立及应用[J].中国兽医学报,2015,35(7):1056-1059.
[5]DIMITROV K M,RAMEY A M,QIU X,et al.Temporal,geographic,and host distribution of avian paramyxovirus 1(Newcastle disease virus)[J].Infect Gene Evol,2016,39:22-34.
[6]NATH B,BARMAN N N,KUMAR S.Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-2015[J].Microbial Pathog,2016,91:85-91.
[7]SAMADI S,KIANIZADEH M,NAJAFI M F,et al.Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran[J].Virus Gene,2014,48(2):290-295.
[8]丁壮,王承宇,向华,等.鹅副黏病毒分离株生物学特性的研究[J].中国预防兽医学报,2002,24(5):390-391.
[9]ALEXANDER D.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Tech,2000,19(2):443-455.
[10]丁壮,尹仁福,薛聪,等.新城疫流行病学新特点及鹅新城疫防控策略[J].中国兽医学报,2015,35(1):159-168.
[11]HU S,MA H,WU Y,et al.A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics[J].Vaccine,2009,27(6):904-910.
[12]CHONG Y L,PADHI A,HUDSON P J,et al.The effect of vaccination on the evolution and population dynamics of avian paramyxovirus-1[J].PLoS Pathog,2010,6(4):e1000872.