牛结节性皮肤病病毒野毒株TaqMan-MGB荧光定量PCR检测方法的建立
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摘要
为鉴别牛结节性皮肤病病毒野毒株(wild type lumpy skin disease virus,WT LSDV),本研究根据GenBank中发表的WT LSDV全基因组序列,设计并合成1对特异性引物和MGB探针,优化反应条件,建立了鉴别检测WT LSDV的TaqMan-MGB荧光定量PCR方法。结果显示,该方法仅对WT LSDV核酸有特异性扩增,对LSDV疫苗株等其他病毒核酸无交叉反应;标准曲线在一定浓度范围内呈现良好的线性关系,标准曲线为y=-3.361 x+34.979,R~2>0.999,扩增效率为98.39%,最低检测限为4.6 copies/μL。组内、组间重复性试验的变异系数均小于3%。对63份模拟临床样品的检测结果显示,本研究建立荧光定量PCR方法的阳性检出率为90.57%,OIE推荐的普通PCR方法的阳性检出率为64.15%,两种方法检测临床样品均为WT LSDV核酸阴性。结果表明,建立的WT LSDV TaqMan-MGB荧光定量PCR方法特异、敏感、快速、重复性好,且能快速准确地检测WT LSDV,适用于临床样品的鉴别检测,为该病的鉴别、监测、防控提供了技术支撑。
In order to indentificate wild type lumpy skin disease virus(WT LSDV),the real-time PCR was established with the specific primers and TaqMan MGB probes designed according to the conserved gene sequences of wild LSDV.The reaction conditions,specificity and sensitivity of this method were optimized.The assay was highly specific and sensitive with the detection limit of 4.6 copies/μ L for WT LSDV,but no amplication for YM LSDV,SPPV,GTPV,BTV,PPRV,cowpox virus,and swine pox virus.The standard curve exhibited a good linear relationship within a certain concentration range,and the standard curve was y=-3.361 x+34.979,R~2>0.999.The amplification efficiency of the method was up to 98.39%.In addition,the coefficient of variation was less than 3.0% for both intra-assay and inter-assay.Moreover,a clinical test showed that the method could accurately detect the nucleic acids of wild LSDV in clinical samples.The above-mentioned results showed that this real-time PCR can be used as an effective tool for rapid detection and epidemic surveillance of LSDV.
In order to indentificate wild type lumpy skin disease virus(WT LSDV),the real-time PCR was established with the specific primers and TaqMan MGB probes designed according to the conserved gene sequences of wild LSDV.The reaction conditions,specificity and sensitivity of this method were optimized.The assay was highly specific and sensitive with the detection limit of 4.6 copies/μ L for WT LSDV,but no amplication for YM LSDV,SPPV,GTPV,BTV,PPRV,cowpox virus,and swine pox virus.The standard curve exhibited a good linear relationship within a certain concentration range,and the standard curve was y=-3.361 x+34.979,R~2>0.999.The amplification efficiency of the method was up to 98.39%.In addition,the coefficient of variation was less than 3.0% for both intra-assay and inter-assay.Moreover,a clinical test showed that the method could accurately detect the nucleic acids of wild LSDV in clinical samples.The above-mentioned results showed that this real-time PCR can be used as an effective tool for rapid detection and epidemic surveillance of LSDV.
引文
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[22] MENASHEROW S,RUBINSTEIN GIUNI M,KOVTUNENKO A,et al.Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus(LSDV)[J].Journal of Virological Methods,2014,199(4):95-101.
[23] EIRINI I A,SERAFEIM C CH,ANDY H,et al.Development and validation of a TaqMan probe-based real-time PCR method for the differenti ation of wild type lumpy skin disease virus from vac cine virus strains[J].Journal of Virological Methods,2017,249(11):48-57.
[24] VIDANOVIC D,SEKLER M,PETROVIC T,et al.Real-time PCR assays for the species detection of field Balkan strains of lumpy skin disease virus[J].Acta Veterinaria,2016,66(4):444-454.
[2] European Food Safety Authority(EFSA).Urgent advice on lumpy skin disease EFSA panel on animal health and welfare[J].European Food Safety Authority Journal,2016,14(8):4573.
[3] CARN V M.Control of capripoxvirus infections[J].Vaccine,1993,11(13):1275-1279.
[4] BABIUK S,BOWDEN TR,PARKYN G,et al.Yemen and vietnam capripoxviruses demonst rate a distinct host preference for goats compared with sheep[J].The Journal of General Virology,2009,90(1):105-114.
[5] DAVIES F G.Lumpy skin disease,an African capripoxviruses disease of cattle[J].British Veterinary Journal,1991,147(6):489-503.
[6] NEAMATALLAH A N F.Immunological,hematological,biochemical,and histopathological studies on cows naturally infected with lumpy skin disease[J].Veterinary World,2015,8(9):1131-1136.
[7] WAINWRIGHT S,El IDRISSI A,MATTIOLI R,et al.Emergence of lumpy skin disease in the Eastern Mediterranean Basin countries[J].FAO Empres Watch,2013,29(12):1-6.
[8] European Food Safety Authority(EFSA).Scientific opinion on lumpy skin disease[J].European Food Safety Authority Journal,2015,13(1):3986.
[9] TASIOUDI,K E,ANTONIOU S E,ILIADOU P,et al.Emergence of lumpy skin disease in Greece,2015[J].Transboundary and Emerging Diseases,2016,63(3):260-265.
[10] TULMAN E R,AFONSO C L,LU Z, et al.Genome of lumpy skin disease virus[J].Journal of Virology,2001,75(15):7122-7130.
[11] TULMAN E R,AFONSO C L,LUZ,et al.The genomes of sheeppox and goatpox viruses[J].Journal of Virology,2002,76(12):6054-6061.
[12] BENGERA J,KLEMENT E,KHINICH E,et al.Comparison of the efficacy of neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin diseasethe results of a randomized controlled field study[J].Vaccine,2015,33(38):4837-4842.
[13] AGIANNIOTAKI E I,TASIOUDI K E,CHAINTOUTIS S C,et al.Lumpy skin disease outbreaks in Greece during 2015—2016,implementation of emergency immunization and genetic differentiation between field isolates and vaccine virus strains[J].Veterinary Microbiology,2017,201(3):78-84.
[14] CARAGUEL C G B,STRYHN H,GAGNE N,et al.Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose:analytical and epidemiological approaches[J].Journal of Veterinary Diagnostic Investigation,2011,23(1):2-15.
[15] TUPPURAINEN E S,VENTER E H,COETZER J A W.The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques[J].The Onderstepoort Journal of Veterinary Research,2005,72(2):153-164.
[16] EIRINI I,AGIANNIOTAKI,SERAFEIM C,et al.Development and validation of a TaqMan probe-based realtime PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains[J].Journal of Virological Methods,2017,249(8):48-57.
[17] KITCHING R P.Vaccines for lumpy skin disease,sheep pox and goat pox[J].Developments in Biologicals,2003,114(1):161-167.
[18] ORLOVA E S,SHCHERBAKOV A V,DIEV V I,et al.Differentiation of capripoxvirus species and strains by polymerase chain reaction[J].Molecular Biology,2006,40(1):139-145.
[19] CHARLES E L,CHRISTIAN L G,ROLAND S,et al.Use of the Capripoxvirus homologue of vaccinia virus 30kDa RNA polymerase subunit(RPO30)gene as a novel diagnostic and genotyping target:development of a classical PCR method to differentiate goat poxvirus from sheep poxvirus[J].Vet erinary Microbiology,2010,149(1-2):30-39.
[20] CHARLES E L,MAMADOU L,WILFRIED G,et al.Real-time PCR method for simultaneous detection,quantitation and differentiation of capripoxviruses[J].Journal of Virological Methods,2010,171(1):134-140.
[21]聂福平,王昱,黄秋华,等.牛结节性皮肤病病毒TaqMan-MGB荧光PCR检测方法的建立[J].中国兽医科学,2017,47(9):1129-1134.NIE Fu-ping,WANG Yu,HUANG Qiu-hua,et al.Establishment of TaqMan-MGB real-time fluorescent PCR for rapid detection of lumpy skin disease virus[J].Chinese Veterinary Science,2017,47(9):1129-1134.
[22] MENASHEROW S,RUBINSTEIN GIUNI M,KOVTUNENKO A,et al.Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus(LSDV)[J].Journal of Virological Methods,2014,199(4):95-101.
[23] EIRINI I A,SERAFEIM C CH,ANDY H,et al.Development and validation of a TaqMan probe-based real-time PCR method for the differenti ation of wild type lumpy skin disease virus from vac cine virus strains[J].Journal of Virological Methods,2017,249(11):48-57.
[24] VIDANOVIC D,SEKLER M,PETROVIC T,et al.Real-time PCR assays for the species detection of field Balkan strains of lumpy skin disease virus[J].Acta Veterinaria,2016,66(4):444-454.