火鸡疱疹病毒荧光定量PCR检测方法的建立及初步应用
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摘要
本研究针对火鸡疱疹病毒(Herpesvirus of turkey,HVT)FC-126毒株sorf1基因序列,设计特异性的扩增引物和探针,建立了检测HVT的TaqMan荧光定量PCR方法,并对其特异性、敏感性和重复性进行检测。结果表明,建立的荧光定量PCR方法灵敏度高,最低可检测到1×10~2 copies/μL,比普通PCR灵敏10倍;对鸭瘟病毒、传染性喉气管炎病毒、鹅细小病毒、4型禽腺病毒和8型禽腺病毒均无特异性扩增,具有较好的特异性;组间和组内变异系数均小于1%,表明该方法重复性好。用建立的荧光定量PCR方法对82份HVT样品进行检测,结果43份为阳性,普通PCR只检测出30份阳性样品。结果表明本研究建立的HVT TaqMan荧光定量PCR检测方法特异性好,灵敏度高,重要性好,为监测HVT的病毒含量提供了一种有效的手段。
A pair of speci?c primers and a probe were designed for development of a ?uorescence quantitative PCR according to the sorf 1 gene sequence of Herpesvirus of turkey(HVT) FC-126 strain. The results demonstrated that this PCR assay was more sensitive for HVT with a detection limit of 1×10~2 copies/μL, which was 10-?od higher than conventional PCR. The assay also proved to be speci?c without ampli?cation from Duck plague virus(DPV), Infectious laryngotracheitis virus(ILTV), Goose parvovirus(GPV), Fowl adenovirus serotype 4(FAdV-4) and Fowl adenovirus serotype 8(FAdV-8). The variabilities of inter-and intra-assay trials were less than1%, indicating a good reproducibility. Subsequently, this ?uorescent quantitative PCR was used to test clinical samples. As a result, 43 out of 82 samples were HVT positive in the ?uorescent quantitative PCR while 30 samples were HVT positive in conventional PCR.Therefore, the ?uorescent quantitative PCR developed in this study was highly speci?c and sensitive for detection of HVT.
A pair of speci?c primers and a probe were designed for development of a ?uorescence quantitative PCR according to the sorf 1 gene sequence of Herpesvirus of turkey(HVT) FC-126 strain. The results demonstrated that this PCR assay was more sensitive for HVT with a detection limit of 1×10~2 copies/μL, which was 10-?od higher than conventional PCR. The assay also proved to be speci?c without ampli?cation from Duck plague virus(DPV), Infectious laryngotracheitis virus(ILTV), Goose parvovirus(GPV), Fowl adenovirus serotype 4(FAdV-4) and Fowl adenovirus serotype 8(FAdV-8). The variabilities of inter-and intra-assay trials were less than1%, indicating a good reproducibility. Subsequently, this ?uorescent quantitative PCR was used to test clinical samples. As a result, 43 out of 82 samples were HVT positive in the ?uorescent quantitative PCR while 30 samples were HVT positive in conventional PCR.Therefore, the ?uorescent quantitative PCR developed in this study was highly speci?c and sensitive for detection of HVT.
引文
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[9]吕让,毛雅元,杨国辉,等.禽流感新型疫苗研究进展[J].中国畜牧兽医,2015,42(6):1608-1612.
[10]Tang N,Zhang Y,Pedrera M,et al.A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Casq system[J].Vaccine,2018,36(5):716-722.
[2]高宏博,崔红玉,石星明,等.表达H5亚型禽流感病毒HA基因重组火鸡疱疹病毒的构建[J].中国预防兽医学报,2010,32(5):329-333.
[3]李奇蒙,陈普成,柳金雄,等.表达传染性法氏囊病毒VP2蛋白的重组火鸡疱疹病毒的构建[J].中国预防兽医学报,2014,36(7):515-518.
[4]Gimeno I M,Cortes A L,Faiz N,et al.Efficacy of various HVT vaccines(conventional and recombinant)against Marek's disease in broiler chickens:effect of dose and age of vaccination[J].Avian Dis,2016,60(3):662-668.
[5]潘群兴,毕振威,王永山,等.表达IBDV AH1株VP2嵌合法氏囊素三肽基因的重组火鸡疱疹病毒的构建及免疫功能分析[J].中国兽医科学,2017,47(9):1073-1079.
[6]康少敏.鸡马立克氏病的预防和治疗[J].中兽医学杂志,2017(6):58.
[7]徐文财,刘秋,秦爱建,等.马立克氏病病毒RB1B株UL14蛋白原核表达及多抗血清制备[J].中国动物传染病学报,2013,21(3):8-12.
[8]兰德松.火鸡疱疹病毒细菌人工染色体及重组火鸡疱疹病毒rHVT-HA的构建[D].北京:中国农业科学院,2008.
[9]吕让,毛雅元,杨国辉,等.禽流感新型疫苗研究进展[J].中国畜牧兽医,2015,42(6):1608-1612.
[10]Tang N,Zhang Y,Pedrera M,et al.A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Casq system[J].Vaccine,2018,36(5):716-722.