毛皮兽阿留申病毒SYBR Green Ⅰ-qPCR检测方法的建立及病毒种鉴别
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摘要
为建立快速定量检测、鉴别毛皮兽阿留申病毒的SYBR Green ⅠqPCR方法,以阿留申病毒属内病毒序列为参考,在VP2序列高变区的保守区段设计合成1对引物。结果,该方法检测水貂阿留申病毒(AMDV)、狐貉阿留申病毒(RF AV)的最低浓度分别为5.38×10~2copies/μL、5.93×10~2copies/μL,且通过熔解曲线可区分鉴别不同病毒种。对55份临床样品以及4株RFAV进行定量检测,结果,有13份PCR检测为阴性的样品被qPCR检测为阳性,说明qPCR检测方法的灵敏度优于普通PCR。上述结果表明,本试验建立的方法是一种高敏感性、特异性检测毛皮兽阿留申病毒,并可通过实测Tm值区分阿留申病毒的分子诊断技术。
To establish a rapid quantitative detection and identification method,SYBR Green I q PCR for fur animal amdoparvovirus,a pair of primers was designed and synthesized.It was located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.In result,the minimum concentrations of Aleutian mink disease virus(AMDV) and raccoon dog and arctic fox amdoparvovirus(RFAV) were 5.38×10~2 copies/μL and 5.93×10~2 copies/μL respectively,and the virus was clearly distinguished by the melt curve.The q PCR testing of 55 clinical specimens and 4 strains of RFAV have been conducted.It was found that 13 samples were negative in PCR but positive in q PCR,indicating that the sensitivity of q PCR is superior to ordinary PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which can be used to clearly distinguish different amdoparvoviruses by measuring Tm of viral amplicon.
To establish a rapid quantitative detection and identification method,SYBR Green I q PCR for fur animal amdoparvovirus,a pair of primers was designed and synthesized.It was located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in Gen Bank.In result,the minimum concentrations of Aleutian mink disease virus(AMDV) and raccoon dog and arctic fox amdoparvovirus(RFAV) were 5.38×10~2 copies/μL and 5.93×10~2 copies/μL respectively,and the virus was clearly distinguished by the melt curve.The q PCR testing of 55 clinical specimens and 4 strains of RFAV have been conducted.It was found that 13 samples were negative in PCR but positive in q PCR,indicating that the sensitivity of q PCR is superior to ordinary PCR.In conclusion,the method has a high sensitivity and specific detection of fur animal amdoparvovirus,which can be used to clearly distinguish different amdoparvoviruses by measuring Tm of viral amplicon.
引文
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[13]李艳伍.貂阿留申病病毒流行毒株分子特征及其VP2基因重组杆状病毒免疫特性研究[D].南京:南京农业大学,2012.LI Yan-wu.Molecular characterization of mink Aleutian disease virus endemic strains and immunicity of recombinanted VP2 protein expressed in baculoviruses[D].Nanjing:Nanjing Agricultural University,2012.(in Chinese)
[14]王心舞,冷雪,杜锐.不同毒力水貂阿留申病毒在猫肾细胞中的增殖特性及凋亡特性的比较研究[J].中国畜牧兽医,2017,44(9):2783-2791.WANG Xin-wu,LENG Xue,DU Rui.Comparative study of proliferation and apoptosis characteristics of different virulence of Aleutian mink disease virus in CRFK cells[J].Chinese Animal Husbandry and Veterinary Medicine,2017,44(9):2783-2791.(in Chinese)
[15]滕传杰.水貂阿留申疾病的双重PCR检测方法建立及其应用[D].泰安:山东农业大学,2018.TENG Chuan-jie.The development and application of a duplex PCR method for the detection of Aleutian mink disease[D].Tai′an:Shandong Agricultural University,2018.(in Chinese)
[16]ZHANG Z,WANG B,HU S,et al.Detection of Aleutian disease virus by loop-mediated isothermal amplification[J].Virus Disease,2015,26(3):203-206.
[17]刘艺,冷雪,时坤,等.水貂阿留申病毒检测方法研究进展[J].动物医学进展,2018,39(10):74-77.LIU Yi,LENG Xue,SHI Kun,et al.Progress on detection method of Aleutian disease virus[J].Progress in Veterinary Medicine,2018,39(10):74-77.(in Chinese)
[18]庄金秋,梅建国,王玉茂,等.水貂阿留申病毒最新检测方法研究进展[J].现代畜牧兽医,2017(11):53-58.ZHUANG Jin-qiu,MEI Jian-guo,WANG Yu-mao,et al.Research progress on the detection methods of Aleutian disease of mink[J].Modern Journal of Animal Husbandry and Veterinary,2017(11):53-58.(in Chinese)
[19]CHO H J,GREENFIELD J.Eradication of Aleutian disease of mink by eliminating positive counterimmunoelectrophoresis test reactors[J].Journal of Clinical Microbiology,1978,7(1):18-22.
[20]QIU J,CHENG F,BURGER L R,et al.The transcription profile of Aleutian mink disease virus in CRFKcells is generated by alternative processing of pre-mRNAs produced from a single promoter[J].Journal of Virology,2006,80(2):654-662.
[2]LI L,PESAVENTO P A,WOODS L,et al.Novel amdovirus in gray foxes[J].Emerging Infectious Diseases,2011,17(10):1876-1878.
[3]BODEWES R,RUIZ-GONZALEZ A,SCHAPENDONK C M,et al.Viral metagenomic analysis of feces of wild small carnivores[J].Virology Journal,2014,11:89.
[4]SHAO X Q,WEN Y J,BA H X,et al.Novel amdoparvovirus infecting farmed raccoon dogs and arctic foxes[J].Emerging Infectious Diseases,2014,20(12):2085-2088.
[5]CANUTI M,DOYLE H E,BRITTON A P,et al.Full genetic characterization and epidemiology of a novel amdoparvovirus in striped skunk(Mephitis mephitis)[J].Emerging Microbes&Infections,2017,6(5):e30.
[6]ALEX C E,KUBISKI S V,LI L,et al.Amdoparvovirus infection in red pandas(Ailurus fulgens)[J].Veterinary Pathology,2018,55(4):552-561.
[7]邵西群,章秀婷,于淼,等.貉狐源阿留申病病毒的感染检测与基因特征[J].中国兽医科学,2015,45(6):589-595.SHAO Xi-qun,ZHANG Xiu-ting,YU Miao,et al.Detection of raccoon dog and fox Amdoparvovirus infection and viral genetic characteristics[J].Chinese Veterinary Science,2015,45(6):589-595.(in Chinese)
[8]PRIETO A,DIAZ-CAO J M,FERNANDEZ-ANTONIO R,et al.Application of real-time PCR to detect Aleutian mink disease virus on environmental farm sources[J].Veterinary Microbiology,2014,173(3-4):355-359.
[9]PRIETO A,FERNANDEZ-ANTONIO R,DIAZ-CAO J M,et al.Distribution of Aleutian mink disease virus contamination in the environment of infected mink farms[J].Veterinary Microbiology,2017,204:59-63.
[10]KOWALCZYK M,JAKUBCZAK A,HORECKA B,et al.A comparative molecular characterization of AMDVstrains isolated from cases of clinical and subclinical infection[J].Virus Genes,2018,54(4):561-569.
[11]PRIETO A,DIAZ-CAO J M,FERNANDEZ-ANTONIO R,et al.Lesser housefly(Fannia canicularis)as possible mechanical vector for Aleutian mink disease virus[J].Veterinary Microbiology,2018,221:90-93.
[12]张瑞.水貂阿留申病毒实时定量PCR检测方法的建立、全基因分析及VP2基因主要功能区的原核表达与鉴定[D].郑州:河南农业大学,2010.ZHANG Rui.Establishment of real-time PCR assay for detecting Aleutian mink disease virus、analysis for its entire gene and prokaryotic expression and identification for the main regions of VP2 gene[D].Zhengzhou:Henan Agricultural University,2010.(in Chinese)
[13]李艳伍.貂阿留申病病毒流行毒株分子特征及其VP2基因重组杆状病毒免疫特性研究[D].南京:南京农业大学,2012.LI Yan-wu.Molecular characterization of mink Aleutian disease virus endemic strains and immunicity of recombinanted VP2 protein expressed in baculoviruses[D].Nanjing:Nanjing Agricultural University,2012.(in Chinese)
[14]王心舞,冷雪,杜锐.不同毒力水貂阿留申病毒在猫肾细胞中的增殖特性及凋亡特性的比较研究[J].中国畜牧兽医,2017,44(9):2783-2791.WANG Xin-wu,LENG Xue,DU Rui.Comparative study of proliferation and apoptosis characteristics of different virulence of Aleutian mink disease virus in CRFK cells[J].Chinese Animal Husbandry and Veterinary Medicine,2017,44(9):2783-2791.(in Chinese)
[15]滕传杰.水貂阿留申疾病的双重PCR检测方法建立及其应用[D].泰安:山东农业大学,2018.TENG Chuan-jie.The development and application of a duplex PCR method for the detection of Aleutian mink disease[D].Tai′an:Shandong Agricultural University,2018.(in Chinese)
[16]ZHANG Z,WANG B,HU S,et al.Detection of Aleutian disease virus by loop-mediated isothermal amplification[J].Virus Disease,2015,26(3):203-206.
[17]刘艺,冷雪,时坤,等.水貂阿留申病毒检测方法研究进展[J].动物医学进展,2018,39(10):74-77.LIU Yi,LENG Xue,SHI Kun,et al.Progress on detection method of Aleutian disease virus[J].Progress in Veterinary Medicine,2018,39(10):74-77.(in Chinese)
[18]庄金秋,梅建国,王玉茂,等.水貂阿留申病毒最新检测方法研究进展[J].现代畜牧兽医,2017(11):53-58.ZHUANG Jin-qiu,MEI Jian-guo,WANG Yu-mao,et al.Research progress on the detection methods of Aleutian disease of mink[J].Modern Journal of Animal Husbandry and Veterinary,2017(11):53-58.(in Chinese)
[19]CHO H J,GREENFIELD J.Eradication of Aleutian disease of mink by eliminating positive counterimmunoelectrophoresis test reactors[J].Journal of Clinical Microbiology,1978,7(1):18-22.
[20]QIU J,CHENG F,BURGER L R,et al.The transcription profile of Aleutian mink disease virus in CRFKcells is generated by alternative processing of pre-mRNAs produced from a single promoter[J].Journal of Virology,2006,80(2):654-662.