PEDV、TGEV、PDCoV、PRoV多重TaqMan荧光定量RT-PCR检测方法的建立及应用
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摘要
为建立鉴别检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)和猪轮状病毒(PRoV)的方法,本研究分别针对PEDV N基因、TGEV M基因、PDCoV M基因和PRoV VP6基因设计特异性引物和TaqMan探针,经过优化反应条件,建立了同时检测4种病毒的多重TaqMan荧光定量RT-PCR方法。结果显示,该方法仅特异性扩增PEDV、TGEV、PDCoV和PRoV,与猪其它主要病毒无交叉反应,特异性较强;对PEDV、TGEV、PDCoV和PRoV的质粒标准品的最低检出限分别为2.06×10~2拷贝/μL、2.06×10~2拷贝/μL、2.06×10~1拷贝/μL、2.06×10~3拷贝/μL,具有较高敏感性;组内与组间变异系数均小于1.1%,具有良好的重复性。应用该方法和普通多重RT-PCR检测临床采集的243份腹泻病料样品,两者的符合率为96.71%。本研究建立的方法为临床PEDV、TGEV、PDCoV、PRoV的鉴别检测及流行病学调查提供了一种快速、特异、敏感、高效的技术手段。
To develop an assay for differential detection of porcine epidemic diarrhea virus(PEDV), porcine transmissible gastroenteritis virus(TGEV), porcine deltacoronavirus(PDCoV) and porcine rotavirus(PRoV), four pairs of specific primers and TaqMan probes were designed for PEDV N gene, TGEV M gene, PDCoV M gene and PRoV VP6 gene, respectively. After optimizing the reaction conditions, a multiplex TaqMan real-time RT-PCR for simultaneous detection of PEDV, TGEV, PDCoV and PRoV was established. The results showed that the established assay could only specifically detected PEDV, TGEV, PDCoV and PRoV, and no other major porcine viruses was detected. The lowest limit of detection was 2.06 ×10~2 copies/μL for PEDV,2.06×10~2 copies/μL for TGEV, 2.06×10~1 copies/μL for PDCoV and 2.06×10~3 copies/μL for PRoV, indicating this assay is highly sensitive. The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 1.1%. This multiplex TaqMan real-time RT-PCR was used to detect 243 diarrhea samples in comparison to conventional multiplex RT-PCR and the coincidence rate of both assay was 96.71%. The results indicated that the established method could provide a rapid, specific,sensitive and efficient technique for differential detection and epidemiological investigation of PEDV, TGEV, PDCoV and PRoV.
To develop an assay for differential detection of porcine epidemic diarrhea virus(PEDV), porcine transmissible gastroenteritis virus(TGEV), porcine deltacoronavirus(PDCoV) and porcine rotavirus(PRoV), four pairs of specific primers and TaqMan probes were designed for PEDV N gene, TGEV M gene, PDCoV M gene and PRoV VP6 gene, respectively. After optimizing the reaction conditions, a multiplex TaqMan real-time RT-PCR for simultaneous detection of PEDV, TGEV, PDCoV and PRoV was established. The results showed that the established assay could only specifically detected PEDV, TGEV, PDCoV and PRoV, and no other major porcine viruses was detected. The lowest limit of detection was 2.06 ×10~2 copies/μL for PEDV,2.06×10~2 copies/μL for TGEV, 2.06×10~1 copies/μL for PDCoV and 2.06×10~3 copies/μL for PRoV, indicating this assay is highly sensitive. The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 1.1%. This multiplex TaqMan real-time RT-PCR was used to detect 243 diarrhea samples in comparison to conventional multiplex RT-PCR and the coincidence rate of both assay was 96.71%. The results indicated that the established method could provide a rapid, specific,sensitive and efficient technique for differential detection and epidemiological investigation of PEDV, TGEV, PDCoV and PRoV.
引文
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[5]Lin Chun-ming,Saif L J,Marthaler D,et al.Evolution,antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains[J].Virus Res,2016,226:20-39.
[6]Zhang Jian-qiang.Porcine deltacoronavirus:Overview of infection dynamics,diagnostic methods,prevalence and genetic evolution[J].Virus Res,2016,226:71-84.
[7]王睿敏,施开创,黎宗强,等.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[J].中国兽医科学,2019,49(02):190-198.
[8]Zhang Qian,Hu Rui-ming,Tang Xi-biao,et al.Occurrence and investigation of enteric viral infections in pigs with diarrhea in China[J].Arch Virol,2013,158(8):1631-1636.
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[10]Seong H K,In J K,Hyum M P,et al.Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].J Virol Methods,2007,146(1-2):172-177.
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[13]刘邓,贾泽颖,张丹,等.猪流行性腹泻病毒、传染性胃肠炎病毒和轮状病毒多重实时荧光定量RT-PCR检测方法的建立[J].黑龙江畜牧兽医,2017,(08):45-50,288-289.
[14]李军,蔡良语,陈兵,等.猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒A多重荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2018,40(9):801-805.
[15]罗尚星,范京惠,刘宝京,等.猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(4):852-858.
[16]张利卫,曹贝贝,李炳晓,等.新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR GreenⅠ双重荧光RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(4):618-624.
[17]陈小金.PEDV、TGEV、Po RV SYBR GreenⅠ实时荧光PCR诊断方法的探索[D].北京:中国农业科学院,2010.
[18]吴洋.猪腹泻相关病毒多重SYBR GreenⅠ荧光定量PCR检测方法的建立及初步应用[D].哈尔滨:东北农业大学,2014.
[19]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.
[20]任玉鹏,张斌,汤承,等.同时检测PEDV和TGEV及PD-CoV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.
[2]Vlasova A N,Amimo J O,Saif L J.Porcine rotaviruses:epidemiology,immune responses and control strategies[J].Viruses,2017,9(3).pii:E48.
[3]Li Wen-tao,Li Heng,Liu Yun-bo,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerg Infect Dis,2012,18(8):1350-1353.
[4]Niederwerder 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].Trans Emerg Dis,2018,65(3):660-675.
[5]Lin Chun-ming,Saif L J,Marthaler D,et al.Evolution,antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains[J].Virus Res,2016,226:20-39.
[6]Zhang Jian-qiang.Porcine deltacoronavirus:Overview of infection dynamics,diagnostic methods,prevalence and genetic evolution[J].Virus Res,2016,226:71-84.
[7]王睿敏,施开创,黎宗强,等.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[J].中国兽医科学,2019,49(02):190-198.
[8]Zhang Qian,Hu Rui-ming,Tang Xi-biao,et al.Occurrence and investigation of enteric viral infections in pigs with diarrhea in China[J].Arch Virol,2013,158(8):1631-1636.
[9]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):216-220.
[10]Seong H K,In J K,Hyum M P,et al.Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].J Virol Methods,2007,146(1-2):172-177.
[11]侯月娥,伍建敏,李中圣.PEDV和TGEV双重Taq Man荧光定量一步法RT-PCR检测方法的建立及应用[J].中国动物传染病学报,2017,25(1):19-25.
[12]许梦怡.猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒多重荧光RT-PCR检测方法的建立[D].江苏:扬州大学,2016.
[13]刘邓,贾泽颖,张丹,等.猪流行性腹泻病毒、传染性胃肠炎病毒和轮状病毒多重实时荧光定量RT-PCR检测方法的建立[J].黑龙江畜牧兽医,2017,(08):45-50,288-289.
[14]李军,蔡良语,陈兵,等.猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒A多重荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2018,40(9):801-805.
[15]罗尚星,范京惠,刘宝京,等.猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(4):852-858.
[16]张利卫,曹贝贝,李炳晓,等.新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR GreenⅠ双重荧光RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(4):618-624.
[17]陈小金.PEDV、TGEV、Po RV SYBR GreenⅠ实时荧光PCR诊断方法的探索[D].北京:中国农业科学院,2010.
[18]吴洋.猪腹泻相关病毒多重SYBR GreenⅠ荧光定量PCR检测方法的建立及初步应用[D].哈尔滨:东北农业大学,2014.
[19]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.
[20]任玉鹏,张斌,汤承,等.同时检测PEDV和TGEV及PD-CoV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.
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