鸭坦布苏病毒RT-PCR检测方法的建立及应用
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摘要
为建立一种能快速检测鸭坦布苏病毒(Tembusu virus,TMUV)的分子生物学方法,从GenBank中下载了51个具有代表性的黄病毒全基因组序列及白洋淀病毒等5个禽源黄病毒株E基因序列,进行了黄病毒E基因序列分析,应用Primer 5.0软件设计了一对引物,扩增目的片段为549 bp,进行了TMUV RT-PCR检测方法的特异性、敏感性、重复性试验,建立了TMUV RT-PCR检测方法应用该方法对收集的14份临床疑似蛋鸭鸭坦布苏病毒病发病样品进行了检测,共检出6份TMUV核酸阳性样品,检出率达42.86%。结果表明,该方法可用于水禽坦布苏病毒病的临床快速诊断、检测及流行病学调查。
To provide a molecular biological method for rapid detection of the duck Tembusu virus( TMUV),the complete genome sequence of 51 representative Flavivirus,E gene of the BYD virus and 5 Avian Flavivirus were downloaded from the GenBank,multiple sequences analysis of Flavivirus E gene was carried out,and using the Primer 5.0 software,a pair of primers were designed,the motive fragment of amplification was 549 bp. A Tembusu virus RT-PCR detective method was established. The method had some advantages such as fast,specific,sensitive and repeatable.The method was used to detect 14 suspected clinic samples. There were 6 TMUV positive samples,the detection rate was 42.86%. The results showed that this method could be used for the rapid diagnosis,detectand epidemiological investigation of the waterfowl Tembusu virus disease.
To provide a molecular biological method for rapid detection of the duck Tembusu virus( TMUV),the complete genome sequence of 51 representative Flavivirus,E gene of the BYD virus and 5 Avian Flavivirus were downloaded from the GenBank,multiple sequences analysis of Flavivirus E gene was carried out,and using the Primer 5.0 software,a pair of primers were designed,the motive fragment of amplification was 549 bp. A Tembusu virus RT-PCR detective method was established. The method had some advantages such as fast,specific,sensitive and repeatable.The method was used to detect 14 suspected clinic samples. There were 6 TMUV positive samples,the detection rate was 42.86%. The results showed that this method could be used for the rapid diagnosis,detectand epidemiological investigation of the waterfowl Tembusu virus disease.
引文
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[11]王林川,李庆阳,陈瑞爱,等.鸭出血性卵巢炎广东病例报道及其RT-PCR方法的建立[J].广东畜牧兽医科技,2011,36(6):27-29.Wang L C,Li Q Y,Chen R A,et al. Duck hemorrhagic ovaritis in Guangdong province and the construction of a RT-PCR detection method[J]. Guangdong Animal Husbandry and Veterinary Medcine Science,2011,36(6):27-29.
[2]范根成,刘东,栾栋祖,等.一种新发鸭病的流行病学调查报告[J].中国动物检疫,2011,28(1):68-69.Fan G C,Liu D,Luan D Z,et al. An epidemiology survey of an new duck disease[J]. Chinese Journal of Animal Quarantine,2011,28(1):68-69.
[3]滕巧泱,颜丕熙,张旭,等.一种新的黄病毒导致蛋鸭产蛋下降及死亡[J].中国动物传染病学报,2010,18(6):1-4.Teng Q Y,Yan P X,Zhang X,et al. An novel flavivirus causing duck egg drops and death[J]. Chinese Journal of Veterinary Parasitology,2010,18(6)1-4.
[4]张大丙.鸭出血性卵巢炎发生的本质与危害[J].中国禽业导刊,2012,29(6):8~9.Zhang D B. Essence and damage of duck hemorrhagic otis[J].Guide of Chinese Poultry,2012,29(6):8-9.
[5]韦天超,农海连,磨美兰,等.鸭出血性卵巢炎病原学研究进展[J].广西畜牧兽医,2012,28(3):182-184.Wei T C,Nong H L,Mo M L,et al. Research progress on etiology of duck hemorrhagic otis[J]. Guangxi Journal of Animal Husbandry and Veterinary Medcine,2012,29(3):182-184.
[6]曹贞贞,张存,黄瑜,等.鸭出血性卵巢炎的初步研究[J].中国兽医杂志,2010,46(12):3-7.Huang Z Z,Zhang C,Huang Y,et al. Preliminary studies on duck hemorrhagic ovaritis[J]. Chinese Journal of Veterinary Medicine,2010,46(12):3-7.
[7]李泽君.鸭坦布苏病毒病病原的分离鉴定及生物学特性研究[J].中国家禽,201l,33(17):34-35.Li Z J. Isolation,identification and biological characteristics of the pathogen of duck tembusu virus disease[J]. China Poultry,2011,33(17):34-35.
[8] Yan P,Zhao Y,Zhang X,et a1. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China[J]. Virology,2011,417(1):1-8.
[9] Lanciotti R S. Molecular Amplification assays for the detection of Flaviviruses[J].Adventure Virus Research,2003,61:67-99.
[10]万春和,施少华,程龙飞,等.鸭出血性卵巢炎病毒RT-PCR检测方法的建立[J].福建农业学报,2011,26(1):10-12.Wan C H,Shi S H,Cheng L F,et al. Establishment of RT-PCR for detecting duck hemorrhagic ovaritis[J]. Fujian Journal of Agricultural Sciences,2011,26(1):67-69.
[11]王林川,李庆阳,陈瑞爱,等.鸭出血性卵巢炎广东病例报道及其RT-PCR方法的建立[J].广东畜牧兽医科技,2011,36(6):27-29.Wang L C,Li Q Y,Chen R A,et al. Duck hemorrhagic ovaritis in Guangdong province and the construction of a RT-PCR detection method[J]. Guangdong Animal Husbandry and Veterinary Medcine Science,2011,36(6):27-29.