传染性法氏囊病病毒RT-LAMP检测方法的建立及其应用
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摘要
为建立一种用于快速检测鸡传染性法氏囊病病毒(IBDV)的逆转录环介导等温扩增技术(RTLAMP)方法,设计了针对VP1基因保守序列的5组引物,采用荧光检测试剂通过实时浊度仪监测阳性扩增结果。结果显示,从5组引物中筛选出了1组对IBDV有效扩增的引物,利用该引物能够在63℃、1 h条件下实现IBDV VP1基因片段的特异性扩增,与其他病毒的核酸无扩增反应;样品RNA的最低检出量为5×10-5ng/L;利用建立的检测方法对14份临床样品进行检测,其阳性检出率为100%。结果表明,建立的IBDV RT-LAMP检测方法具有快速、准确、特异性强、灵敏度高和便捷的特点,可用于IBD临床样品的检测。
To establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)technique for rapid detection of infectious bursal disease virus(IBDV),five groups of primers,including two primer sets,targeting the conserved sequence of VP1 gene,were designed.The positive amplification results were monitored by a real-time turbidimeter using the fluorescence detection reagent.In result,one of the group of primers for efficient amplification of IBDV was screened out from the five primers groups,and the primers were specific in amplification of the IBDV VP1 gene fragment within 1 h at 63 ℃.The primers showed no amplification reaction with nucleic acids of other viruses.And the minimum detectable amount of sample RNA was 5×10-5ng/L.Finaly,fourteen IBD clinical samples were tested using the established assay and the positive detection rate was 100%.The results showed that the established IBDV RT-LAMP detection method is a fast,accurate,specific,sensitive and convenient technique,which can be a potential method for the detection IBDV in clinical IBD samples.
To establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)technique for rapid detection of infectious bursal disease virus(IBDV),five groups of primers,including two primer sets,targeting the conserved sequence of VP1 gene,were designed.The positive amplification results were monitored by a real-time turbidimeter using the fluorescence detection reagent.In result,one of the group of primers for efficient amplification of IBDV was screened out from the five primers groups,and the primers were specific in amplification of the IBDV VP1 gene fragment within 1 h at 63 ℃.The primers showed no amplification reaction with nucleic acids of other viruses.And the minimum detectable amount of sample RNA was 5×10-5ng/L.Finaly,fourteen IBD clinical samples were tested using the established assay and the positive detection rate was 100%.The results showed that the established IBDV RT-LAMP detection method is a fast,accurate,specific,sensitive and convenient technique,which can be a potential method for the detection IBDV in clinical IBD samples.
引文
[1]何秀苗,阳秀英,韦平.传染性法氏囊病及其病原分子生物学研究进展[J].动物医学进展,2007,28(2):48-51.HE Xiu-miao,YANG Xiu-ying,WEI Ping.Progress on infectious bursal disease and the molecular biology of pathogen[J].Progress in Veterinary Medicine,2007,28(2):48-51.(in Chinese)
[2] MULLER H,NITSCHKE R.The two segments of the infectious bursal disease virus genome are circularized by a 90 000-Da protein[J].Virology,1987,159(1):174-177.
[3]张文通,魏凤,苗立中,等.传染性法氏囊病病毒RT-PCR检测及VP2基因序列分析[J].动物医学进展,2014,35(5):8-11.ZHANG Wen-tong,WEI Feng,MIAO Li-zhong,et al. RTPCR detection of infectious bursal disease virus and sequence analysis of its VP2 gene[J].Progress in Veterinary Medicine,2014,35(5):8-11.(in Chinese)
[4]何秀苗,甘珊珊,熊忠贤,等.IBDV荧光定量RT-PCR检测方法的建立及其应用[J].西南农业学报,2013,26(2):798-802.HE Xiu-miao,GAN Shan-shan,XIONG Zhong-xian,et al.Development and application of real time quantitative RT-PCR assay for IBDV detection[J].Southwest China Journal of Agricultural Sciences,2013,26(2):798-802.(in Chinese)
[5]梅永杰.传染性法氏囊病病毒流行株的分离与检测方法的建立[D].南京:南京农业大学,2016.MEI Yong-jie.Isolation of infectious bursal disease virus and development of detection methods[D].Nanjing:Nanjing Agricultural University,2016.(in Chinese)
[6] SONG H S,BAE Y C,PARK S C,et al.Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken[J].Journal of Virological Methods,2018,256:6-11.
[7] XU J T,ZHANG Z M,YIN Y B,et al.Development of reverse-transcription loop-mediated isothermal amplification for the detection of infectious bursal disease virus[J].Journal of Virological Methods,2009,162(1-2):267-271.
[8] WANG Y Q,KANG Z H,GAO H L,et al.A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus[J].Virology Journal,2011,8(1):1-7.
[9] FEMANDEZ-ARIAS A,RISCO C,MARTINEZ S,et al.Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles[J].Journal of General Virology,1998,79(5):1047-1054.
[10]高立,李凯,祁小乐,等.鸡传染性法氏囊病病毒B节段对病毒致病性的影响[J].中国预防兽医学报,2014,36(7):510-514.GAO Li,LI Kai,QI Xiao-le,et al.Segment B of infectious bursal disease virus contributes to viral virulence[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(7):510-514.(in Chinese)
[11] ISLAM M R,RAHMAN S,NOOR M,et al.Differentiation of infectious bursal disease virus(IBDV)genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis[J].Archives of Virology,2012,157(2):333-336.
[12] GHANIEI A,PEIGHAMBARI S M,RAZMYAR J.Identification of very virulent infectious bursal disease viruses by RT-PCR of VP1 gene and speculation about the possible presence of reassortant viruses[J].Journal of Veterinary Research,2011,66(2):153-159.
[13]何秀苗,戴书剑,官丁明,等.近年来我国鸡传染性法氏囊病及其病毒分离株的变化特点[J].广西畜牧兽医,2012,28(2):67-70.HE Xiu-miao,DAI Shu-jian,GUAN Ding-ming,et al.Change characteristics of chicken infectious bursal disease and its virus isolates from China in recent years[J].Guangxi Animal Husbandry&Veterinary Medicine,2012,28(2):67-70.(in Chinese)
[14]杨霖.2011—2012年间IBDV的分离鉴定及不同基因型IBDV流行株的抗原性研究[D].南宁:广西民族大学,2014.YANG Lin.2011—2012 Isolation and identification of IBDV and antigenicity of IBDV strains of different genotypes[D].Nanning:Guangxi University for Nationalities,2014.(in Chinese)
[15]吴禹熹,李璞君,王娟,等.LAMP方法及其在病原微生物检测中的应用[J].中国畜牧兽医,2016,43(2):389-393.WU Yu-xi,LI Pu-jun,WANG Juan,et al.LAMP method and it application in pathogenic microbiological detection[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):389-393.(in Chinese)
[16]吴植,夏文龙,蔡树东,等.鸭坦布苏病毒可视化RTLAMP快速检测方法的建立与初步应用[J].中国畜牧兽医,2017,44(11):3334-3339.WU Zhi,XIA Wen-long,CAI Shu-dong,et al.Establishment and application of visual RT-LAMP for rapid detection of duck tembusu virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3334-3339.(in Chinese)
[17]许传田,鲁梅,胡北侠,等.H9N2亚型禽流感病毒RTLAMP方法建立及临床应用[J].中国兽医学报,2012,32(6):814-817.XU Chuan-tian,LU Mei,HU Bei-xia,et al.Establishment of H9N2 subtypes avian flu virus RT-LAMP methods and its application in large-scale broiler farmers[J].Chinese Journal of Veterinary Science,2012,32(6):814-817.(in Chinese)
[18]刘文俊,黄运茂,阳佑天,等.鹅新城疫病毒RT-LAMP可视化检测方法的建立[J].中国畜牧兽医,2018,45(1):22-31.LIU Wen-jun,HUANG Yun-mao,YANG You-tian,et al.Establishment of RT-LAMP visual detection method for goose newcastle disease virus[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):22-31.(in Chinese)
[19]黄海超,陈轩,杨素,等.禽白血病病毒K亚群RT-LAMP检测方法的建立[J].中国兽医科学,2018,48(2):161-166.HUANG Hai-chao,CHEN Xuan,YANG Su,et al.Development of RT-LAMP method for the detection of avian leukosis virus subgroup K[J].Chinese Veterinary Science,2018,48(2):161-166.(in Chinese)
[20]杨作丰,石霖,邓文超,等.鸡传染性法氏囊病毒反转录环媒恒温检测方法的初步建立[J].现代畜牧兽医,2014(12):12-17.YANG Zuo-feng,SHI Lin,DENG Wen-chao,et al.Rapid detection method preliminary establishment of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2014(12):12-17.(in Chinese)
[21]刘业兵,张磊,宁宜宝,等.猪细小病毒LAMP检测方法的建立[J].中国农业科学,2010,43(14):3012-3018.LIU Ye-bing,ZHANG Lei,NING Yi-bao,et al.Establishment of porcine parvovirus detection by loop-mediated isothermal amplification assay[J].Scientia Agricultura Sinica,2010,43(14):3012-3018.(in Chinese)
[22]陆冰洋,刘华栋,李婷婷,等.传染性法氏囊病病毒RT-LAMP检测方法的建立[J].黑龙江畜牧兽医,2018(15):135-139,243.LU Bing-yang,LIU Hua-dong,LI Ting-ting,et al.Establishment of RT-LAMP detection method for infectious bursal disease virus[J].Heilongjiang Animal Science and Veterinary Medicine,2018(15):135-139,243.(in Chinese)
[23]陈兵,孙洁,陈书琨,等.三种家禽病毒多重RT-LAMP检测方法的建立与应用[J].上海畜牧兽医通讯,2016(1):2-5.CHEN Bing,SUN Jie,CHEN Shu-kun,et al.Establishment and application of multiple RT-LAMP detection methods for three poultry viruses[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2016(1):2-5.(in Chinese)
[24] LEE M S,LIN Y C,LAI G H,et al.One-step reversetranscription loop-mediated isothermal amplification for detection of infectious bursal disease virus[J].Canadian Journal of Veterinary Research,2011,75(2):122-127.
[25] XUE C Y,ZHANG Y,ZHOU Q F,et al.Rapid detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification assay[J].Journal of Veterinary Diagnostic Investigation,2009,21(6):841-843.
[2] MULLER H,NITSCHKE R.The two segments of the infectious bursal disease virus genome are circularized by a 90 000-Da protein[J].Virology,1987,159(1):174-177.
[3]张文通,魏凤,苗立中,等.传染性法氏囊病病毒RT-PCR检测及VP2基因序列分析[J].动物医学进展,2014,35(5):8-11.ZHANG Wen-tong,WEI Feng,MIAO Li-zhong,et al. RTPCR detection of infectious bursal disease virus and sequence analysis of its VP2 gene[J].Progress in Veterinary Medicine,2014,35(5):8-11.(in Chinese)
[4]何秀苗,甘珊珊,熊忠贤,等.IBDV荧光定量RT-PCR检测方法的建立及其应用[J].西南农业学报,2013,26(2):798-802.HE Xiu-miao,GAN Shan-shan,XIONG Zhong-xian,et al.Development and application of real time quantitative RT-PCR assay for IBDV detection[J].Southwest China Journal of Agricultural Sciences,2013,26(2):798-802.(in Chinese)
[5]梅永杰.传染性法氏囊病病毒流行株的分离与检测方法的建立[D].南京:南京农业大学,2016.MEI Yong-jie.Isolation of infectious bursal disease virus and development of detection methods[D].Nanjing:Nanjing Agricultural University,2016.(in Chinese)
[6] SONG H S,BAE Y C,PARK S C,et al.Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken[J].Journal of Virological Methods,2018,256:6-11.
[7] XU J T,ZHANG Z M,YIN Y B,et al.Development of reverse-transcription loop-mediated isothermal amplification for the detection of infectious bursal disease virus[J].Journal of Virological Methods,2009,162(1-2):267-271.
[8] WANG Y Q,KANG Z H,GAO H L,et al.A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus[J].Virology Journal,2011,8(1):1-7.
[9] FEMANDEZ-ARIAS A,RISCO C,MARTINEZ S,et al.Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles[J].Journal of General Virology,1998,79(5):1047-1054.
[10]高立,李凯,祁小乐,等.鸡传染性法氏囊病病毒B节段对病毒致病性的影响[J].中国预防兽医学报,2014,36(7):510-514.GAO Li,LI Kai,QI Xiao-le,et al.Segment B of infectious bursal disease virus contributes to viral virulence[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(7):510-514.(in Chinese)
[11] ISLAM M R,RAHMAN S,NOOR M,et al.Differentiation of infectious bursal disease virus(IBDV)genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis[J].Archives of Virology,2012,157(2):333-336.
[12] GHANIEI A,PEIGHAMBARI S M,RAZMYAR J.Identification of very virulent infectious bursal disease viruses by RT-PCR of VP1 gene and speculation about the possible presence of reassortant viruses[J].Journal of Veterinary Research,2011,66(2):153-159.
[13]何秀苗,戴书剑,官丁明,等.近年来我国鸡传染性法氏囊病及其病毒分离株的变化特点[J].广西畜牧兽医,2012,28(2):67-70.HE Xiu-miao,DAI Shu-jian,GUAN Ding-ming,et al.Change characteristics of chicken infectious bursal disease and its virus isolates from China in recent years[J].Guangxi Animal Husbandry&Veterinary Medicine,2012,28(2):67-70.(in Chinese)
[14]杨霖.2011—2012年间IBDV的分离鉴定及不同基因型IBDV流行株的抗原性研究[D].南宁:广西民族大学,2014.YANG Lin.2011—2012 Isolation and identification of IBDV and antigenicity of IBDV strains of different genotypes[D].Nanning:Guangxi University for Nationalities,2014.(in Chinese)
[15]吴禹熹,李璞君,王娟,等.LAMP方法及其在病原微生物检测中的应用[J].中国畜牧兽医,2016,43(2):389-393.WU Yu-xi,LI Pu-jun,WANG Juan,et al.LAMP method and it application in pathogenic microbiological detection[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):389-393.(in Chinese)
[16]吴植,夏文龙,蔡树东,等.鸭坦布苏病毒可视化RTLAMP快速检测方法的建立与初步应用[J].中国畜牧兽医,2017,44(11):3334-3339.WU Zhi,XIA Wen-long,CAI Shu-dong,et al.Establishment and application of visual RT-LAMP for rapid detection of duck tembusu virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3334-3339.(in Chinese)
[17]许传田,鲁梅,胡北侠,等.H9N2亚型禽流感病毒RTLAMP方法建立及临床应用[J].中国兽医学报,2012,32(6):814-817.XU Chuan-tian,LU Mei,HU Bei-xia,et al.Establishment of H9N2 subtypes avian flu virus RT-LAMP methods and its application in large-scale broiler farmers[J].Chinese Journal of Veterinary Science,2012,32(6):814-817.(in Chinese)
[18]刘文俊,黄运茂,阳佑天,等.鹅新城疫病毒RT-LAMP可视化检测方法的建立[J].中国畜牧兽医,2018,45(1):22-31.LIU Wen-jun,HUANG Yun-mao,YANG You-tian,et al.Establishment of RT-LAMP visual detection method for goose newcastle disease virus[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):22-31.(in Chinese)
[19]黄海超,陈轩,杨素,等.禽白血病病毒K亚群RT-LAMP检测方法的建立[J].中国兽医科学,2018,48(2):161-166.HUANG Hai-chao,CHEN Xuan,YANG Su,et al.Development of RT-LAMP method for the detection of avian leukosis virus subgroup K[J].Chinese Veterinary Science,2018,48(2):161-166.(in Chinese)
[20]杨作丰,石霖,邓文超,等.鸡传染性法氏囊病毒反转录环媒恒温检测方法的初步建立[J].现代畜牧兽医,2014(12):12-17.YANG Zuo-feng,SHI Lin,DENG Wen-chao,et al.Rapid detection method preliminary establishment of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2014(12):12-17.(in Chinese)
[21]刘业兵,张磊,宁宜宝,等.猪细小病毒LAMP检测方法的建立[J].中国农业科学,2010,43(14):3012-3018.LIU Ye-bing,ZHANG Lei,NING Yi-bao,et al.Establishment of porcine parvovirus detection by loop-mediated isothermal amplification assay[J].Scientia Agricultura Sinica,2010,43(14):3012-3018.(in Chinese)
[22]陆冰洋,刘华栋,李婷婷,等.传染性法氏囊病病毒RT-LAMP检测方法的建立[J].黑龙江畜牧兽医,2018(15):135-139,243.LU Bing-yang,LIU Hua-dong,LI Ting-ting,et al.Establishment of RT-LAMP detection method for infectious bursal disease virus[J].Heilongjiang Animal Science and Veterinary Medicine,2018(15):135-139,243.(in Chinese)
[23]陈兵,孙洁,陈书琨,等.三种家禽病毒多重RT-LAMP检测方法的建立与应用[J].上海畜牧兽医通讯,2016(1):2-5.CHEN Bing,SUN Jie,CHEN Shu-kun,et al.Establishment and application of multiple RT-LAMP detection methods for three poultry viruses[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2016(1):2-5.(in Chinese)
[24] LEE M S,LIN Y C,LAI G H,et al.One-step reversetranscription loop-mediated isothermal amplification for detection of infectious bursal disease virus[J].Canadian Journal of Veterinary Research,2011,75(2):122-127.
[25] XUE C Y,ZHANG Y,ZHOU Q F,et al.Rapid detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification assay[J].Journal of Veterinary Diagnostic Investigation,2009,21(6):841-843.
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