猪流行性腹泻病毒猪传染性胃肠炎病毒猪Delta冠状病毒和猪轮状病毒多重RT-PCR检测方法的建立
|
摘要
针对猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪Delta冠状病毒(PDCoV)和猪轮状病毒(PRoV)基因序列,分别设计特异性引物,扩增PEDV N基因(750 bp)、TGEV M基因(544 bp)、PDCoV N基因(183 bp)和PRoV VP6基因(329 bp)。经过对引物浓度、退火温度等反应条件的优化及特异性、敏感性、重复性试验,成功建立了快速鉴别检测PEDV、TGEV、PDCoV、PRoV的多重RT-PCR方法。该方法仅对PEDV、TGEV、PDCoV、PRoV检测为阳性,对口蹄疫病毒(FMDV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)等主要病毒的扩增均为阴性。PEDV、TGEV、PDCoV、PRoV重组质粒标准品的检出下限分别为1.57×10~2、1.57×10~3、1.57×10~2、1.57×10~2copies/L。相同条件下的重复性试验获得均匀一致的结果。应用所建立的方法检测2017—2018年采集自广西各地的270份腹泻病料,结果显示,PEDV、TGEV、PDCoV、PRoV的阳性率分别为15.56%、11.48%、10.74%、1.85%,并且存在混合感染现象。上述结果表明,所建立的多重RT-PCR方法可用于PEDV、TGEV、PDCoV、PRoV的快速鉴别检测及流行病学调查。
To establish a rapid method for simultaneous detection of porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCo V) and porcine rotavirus(PRo V),four pairs of specific primers were designed according to gene sequences for amplifying PEDV N gene(750 bp),TGEV M gene(544 bp),PDCo V N gene(183 bp) and PRo V VP6 gene(329 bp),respectively.After optimizing the primer concentration,annealing temperature and other reaction conditions,a specific,sensitive and reproducible multiplex RT-PCR method was established. The assay could specifically amplify the target fragments from PEDV,TGEV,PDCoV and PRoV,but could not amplify the target fragments from FMDV,PRRSV,CSFV,PRV,PCV2 and PPV.The lowest detection limits of PEDV,TGEV,PDCo V and PRo V were 1.57×10~2,1.57×10~3,1.57×10~2,1.57×10~2copies/L,respectively.The repeatability test underthe same reaction conditions resulted in uniform and consistent results.The established assay was used to detect 270 clinical diarrhea samples collected from Guangxi Province during 2017—2018,and the positive rates of PEDV,TGEV,PDCo V and PRo V were 15.56%,11.48%,10.74% and 1.85%,respectively.There existed co-infections among four viruses.The results indicated that the multiplex RT-PCR for detection of PEDV,TGEV,PDCo V and PRo V was successfully established and could be used for differential detection and epidemiological investigation of these pathogens.
To establish a rapid method for simultaneous detection of porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCo V) and porcine rotavirus(PRo V),four pairs of specific primers were designed according to gene sequences for amplifying PEDV N gene(750 bp),TGEV M gene(544 bp),PDCo V N gene(183 bp) and PRo V VP6 gene(329 bp),respectively.After optimizing the primer concentration,annealing temperature and other reaction conditions,a specific,sensitive and reproducible multiplex RT-PCR method was established. The assay could specifically amplify the target fragments from PEDV,TGEV,PDCoV and PRoV,but could not amplify the target fragments from FMDV,PRRSV,CSFV,PRV,PCV2 and PPV.The lowest detection limits of PEDV,TGEV,PDCo V and PRo V were 1.57×10~2,1.57×10~3,1.57×10~2,1.57×10~2copies/L,respectively.The repeatability test underthe same reaction conditions resulted in uniform and consistent results.The established assay was used to detect 270 clinical diarrhea samples collected from Guangxi Province during 2017—2018,and the positive rates of PEDV,TGEV,PDCo V and PRo V were 15.56%,11.48%,10.74% and 1.85%,respectively.There existed co-infections among four viruses.The results indicated that the multiplex RT-PCR for detection of PEDV,TGEV,PDCo V and PRo V was successfully established and could be used for differential detection and epidemiological investigation of these pathogens.
引文
[1] NIEGERWERDER M C,HESSE R A.Swine enteric coronavirus disease:A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada[J].Transboundary and Emerging Diseases,2018,5(3):660-675.
[2] VLASOVA A N,AMIMO J O,SAIF L J.Porcine rotaviruses:epidemiology,immune responses and control strategies[J].Viruses,2017,9(3):48.
[3] WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus Research,2016,226:7-13.
[4] DONG N,FANG L,ZENG S,et al.Porcine deltacoronavirus in mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[5]韩丽,周雍,李炳晓,等.猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J].中国兽医科学,2017,47(5):557-562.HAN Li,ZHOU Yong,LI Bing-xiao,et al.Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus[J].Chinese Veterinary Science,2016,47(5):557-562.(in Chinese)
[6]任玉鹏,张斌,汤承,等.同时检测PEDV和TGEV及PDCoV的多重RT-PCR方法的建立及初步应用[J].中国兽医科学,2016,46(6):756-762.REN Yu-peng,ZHANG Bin,TANG Cheng,et al.Development and application of a multiplex RT-PCR for detecting PEDV,TGEV and PDCoV[J].Chinese Veterinary Science,2016,46(6):756-762.(in Chinese)
[7]韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(1):11-16.WEI Xue-lei,LIANG Qing-qing,CAO Bei-bei,et al.Establishment and application of multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Chinese Journal of Veterinary Science,2018,38(1):11-16.(in Chinese)
[8]张坤,何启盖.猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A型轮状病毒多重RT-PCR检测方法的建立及临床应用[J].畜牧兽医学报,2010,41(8):1001-1005.ZHANG Kun,HE Qi-gai.Establishment and clinical application of a multiplex reverse transcriptionPCR for detection of porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):1001-1005.(in Chinese)
[9]郭容利,何孔旺,倪艳秀,等.PEDV、TGEV、PARV多重RTPCR检测方法的建立及其应用[J].江苏农业学报,2013,29(5):1065-1069.GUO Rong-li,HE Kong-wang,NI Yan-xiu,et al.Establishment and clinical application of a multiplex reverse transcription-PCR for detection of porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Jiangsu Journal of Agricultural Science,2013,29(5):1065-1069.(in Chinese)
[10]李丽敏,朱卫霞,王晓波,等.猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪A型轮状病毒多重RT-PCR方法的建立及其应用[J].中国兽医学报,2016,36(2):216-221.LI Li-min,ZHU Wei-xia,WANG Xiao-bo,et al.Establishment and application of a multiplex reverse transcription-PCR for detecting porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Chinese Journal of Veterinary Science,2016,36(2):216-221.(in Chinese)
[11]龙飞翔,施开创,张珍,等.PEDV、TGEV和PRoV多重RTPCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(10):2518-2526.LONG Fei-xiang,SHI Kai-chuang,ZHANG Zhen,et al.Establishment and application of a multiple RTPCR assay for detection of PEDV,TGEV and PRoV[J].Chinese Animal Husbandry and Veterinary Medicine,2016,43(10):2518-2526.(in Chinese)
[12]霍金耀,郑逢梅,陈陆,等.猪流行性腹泻病毒、猪传染性胃肠炎病毒、A群轮状病毒和脊病毒多重RT-PCR检测方法的建立及临床应用[J].中国兽医学报,2013,33(12):1792-1797.HUO Jin-yao,ZHENG Feng-mei,CHEN Lu,et al.Establishment and clinical application of a multiplex reverse transcription-PCR for rapid detection of porcine epidemic diarrhea virus,transmissible gastroenteritis virus,group A rotavirus,and porcine kobuvirus[J].Chinese Journal of Veterinary Science,2013,33(12):1792-1797.(in Chinese)
[13] ZHAO J,SHI B J,HUANG X G,et al.A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to2012[J].Journal of Virological Methods,2013,194(1-2):107-112.
[14]熊年年,陶洁,张清真,等.猪四种病毒性腹泻病原多重PCR检测方法的建立及应用[J].中国兽医科学,201646(8):972-978.XIONG Nian-nian,TAO Jie,ZHANG Qing-zhen,et al.Establishment and application of the multiplex PCR for detection of four porcine viral diarrhea viruses[J].Chinese Veterinary Science,2016,46(8):972-978.(in Chinese)
[15]张帆帆,宋德平,周信荣,等.新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J].中国农业科学,2016,49(7):1408-1416.ZHANG Fan-fan,SONG De-ping,ZHOU Xin-rong,et al.Establishment and application of a RT-PCR assay for detection of newly emerged porcine deltacoronavirus[J].Scientia Agricultura Sinica,2016,49(7):1408-1416.(in Chinese)
[16] GRAY J,COUPLAND L J.The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections[J].Epidemiology and Infection,2014,142(1):1-11.
[17]赵津,禹泽忠,冯刚,等.猪病毒病PCV2、PEDV、TGEV、GAR的复合PCR方法建立及应用[J].西南农业学报,2016,29(4):982-987.ZHAO Jin,YU Ze-zhong,FENG Gang,et al.Establishment and application of multiplex RT-PCR for differential diagnosis of four associated diarrhea viruses PCV2,PEDV,TGEV,GAR[J].Southwest China Journal of Agricultural Science,2016,29(4):982-987.(in Chinese)
[18] HU X,LI N,TIAN Z,et al.Molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus HX strain isolated from China[J].BMC Veterinary Research,2015,11(1):72.
[19] LIN C M,GAO X,OKA T,et al.Antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains[J].Journal of Virology,2015,89(6):3332-3342.
[20] ZHANG J.Porcine deltacoronavirus:Overview of infection dynamics,diagnostic methods,prevalence and genetic evolution[J].Virus Research,2016,226:71-84.
[21] SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China:identification,prevalence and fulllength genome sequence analysis[J].Transboundary and Emerging Diseases,2015,62(6):575-580.
[22] LUO J,FANG L,DONG N,et al.Porcine deltacoronavirus(PDCo V)infection suppresses RIG-I-mediated interferon-βproduction[J].Virology,2016,495:10-17.
[2] VLASOVA A N,AMIMO J O,SAIF L J.Porcine rotaviruses:epidemiology,immune responses and control strategies[J].Viruses,2017,9(3):48.
[3] WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus Research,2016,226:7-13.
[4] DONG N,FANG L,ZENG S,et al.Porcine deltacoronavirus in mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[5]韩丽,周雍,李炳晓,等.猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J].中国兽医科学,2017,47(5):557-562.HAN Li,ZHOU Yong,LI Bing-xiao,et al.Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus[J].Chinese Veterinary Science,2016,47(5):557-562.(in Chinese)
[6]任玉鹏,张斌,汤承,等.同时检测PEDV和TGEV及PDCoV的多重RT-PCR方法的建立及初步应用[J].中国兽医科学,2016,46(6):756-762.REN Yu-peng,ZHANG Bin,TANG Cheng,et al.Development and application of a multiplex RT-PCR for detecting PEDV,TGEV and PDCoV[J].Chinese Veterinary Science,2016,46(6):756-762.(in Chinese)
[7]韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(1):11-16.WEI Xue-lei,LIANG Qing-qing,CAO Bei-bei,et al.Establishment and application of multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Chinese Journal of Veterinary Science,2018,38(1):11-16.(in Chinese)
[8]张坤,何启盖.猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A型轮状病毒多重RT-PCR检测方法的建立及临床应用[J].畜牧兽医学报,2010,41(8):1001-1005.ZHANG Kun,HE Qi-gai.Establishment and clinical application of a multiplex reverse transcriptionPCR for detection of porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):1001-1005.(in Chinese)
[9]郭容利,何孔旺,倪艳秀,等.PEDV、TGEV、PARV多重RTPCR检测方法的建立及其应用[J].江苏农业学报,2013,29(5):1065-1069.GUO Rong-li,HE Kong-wang,NI Yan-xiu,et al.Establishment and clinical application of a multiplex reverse transcription-PCR for detection of porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Jiangsu Journal of Agricultural Science,2013,29(5):1065-1069.(in Chinese)
[10]李丽敏,朱卫霞,王晓波,等.猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪A型轮状病毒多重RT-PCR方法的建立及其应用[J].中国兽医学报,2016,36(2):216-221.LI Li-min,ZHU Wei-xia,WANG Xiao-bo,et al.Establishment and application of a multiplex reverse transcription-PCR for detecting porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine group A rotavirus[J].Chinese Journal of Veterinary Science,2016,36(2):216-221.(in Chinese)
[11]龙飞翔,施开创,张珍,等.PEDV、TGEV和PRoV多重RTPCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(10):2518-2526.LONG Fei-xiang,SHI Kai-chuang,ZHANG Zhen,et al.Establishment and application of a multiple RTPCR assay for detection of PEDV,TGEV and PRoV[J].Chinese Animal Husbandry and Veterinary Medicine,2016,43(10):2518-2526.(in Chinese)
[12]霍金耀,郑逢梅,陈陆,等.猪流行性腹泻病毒、猪传染性胃肠炎病毒、A群轮状病毒和脊病毒多重RT-PCR检测方法的建立及临床应用[J].中国兽医学报,2013,33(12):1792-1797.HUO Jin-yao,ZHENG Feng-mei,CHEN Lu,et al.Establishment and clinical application of a multiplex reverse transcription-PCR for rapid detection of porcine epidemic diarrhea virus,transmissible gastroenteritis virus,group A rotavirus,and porcine kobuvirus[J].Chinese Journal of Veterinary Science,2013,33(12):1792-1797.(in Chinese)
[13] ZHAO J,SHI B J,HUANG X G,et al.A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to2012[J].Journal of Virological Methods,2013,194(1-2):107-112.
[14]熊年年,陶洁,张清真,等.猪四种病毒性腹泻病原多重PCR检测方法的建立及应用[J].中国兽医科学,201646(8):972-978.XIONG Nian-nian,TAO Jie,ZHANG Qing-zhen,et al.Establishment and application of the multiplex PCR for detection of four porcine viral diarrhea viruses[J].Chinese Veterinary Science,2016,46(8):972-978.(in Chinese)
[15]张帆帆,宋德平,周信荣,等.新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J].中国农业科学,2016,49(7):1408-1416.ZHANG Fan-fan,SONG De-ping,ZHOU Xin-rong,et al.Establishment and application of a RT-PCR assay for detection of newly emerged porcine deltacoronavirus[J].Scientia Agricultura Sinica,2016,49(7):1408-1416.(in Chinese)
[16] GRAY J,COUPLAND L J.The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections[J].Epidemiology and Infection,2014,142(1):1-11.
[17]赵津,禹泽忠,冯刚,等.猪病毒病PCV2、PEDV、TGEV、GAR的复合PCR方法建立及应用[J].西南农业学报,2016,29(4):982-987.ZHAO Jin,YU Ze-zhong,FENG Gang,et al.Establishment and application of multiplex RT-PCR for differential diagnosis of four associated diarrhea viruses PCV2,PEDV,TGEV,GAR[J].Southwest China Journal of Agricultural Science,2016,29(4):982-987.(in Chinese)
[18] HU X,LI N,TIAN Z,et al.Molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus HX strain isolated from China[J].BMC Veterinary Research,2015,11(1):72.
[19] LIN C M,GAO X,OKA T,et al.Antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains[J].Journal of Virology,2015,89(6):3332-3342.
[20] ZHANG J.Porcine deltacoronavirus:Overview of infection dynamics,diagnostic methods,prevalence and genetic evolution[J].Virus Research,2016,226:71-84.
[21] SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China:identification,prevalence and fulllength genome sequence analysis[J].Transboundary and Emerging Diseases,2015,62(6):575-580.
[22] LUO J,FANG L,DONG N,et al.Porcine deltacoronavirus(PDCo V)infection suppresses RIG-I-mediated interferon-βproduction[J].Virology,2016,495:10-17.