猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用
|
摘要
为了快速、准确地检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome,PRRSV),本研究根据PRRSV基因序列设计特异性引物和探针,建立了一种可同时检测PRRSV经典毒株、高致病性变异毒株以及近几年中国新出现的NADC30-like毒株的TaqMan-MGB实时荧光定量方法,并对该方法的特异性、敏感性和重复性进行验证,同时用建立的实时荧光定量方法与常规PCR方法对临床收集的120份疑似PRRSV样品进行检测。结果表明,该方法特异性良好,对PRRSV的经典毒株、高致病性变异毒株及NADC30-like毒株均有良好的扩增,但对猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)的检测结果均为阴性,无交叉反应;模板浓度在101～108拷贝/μL范围内具有良好的线性关系,标准曲线结果显示其扩增的相关系数为0.9999,扩增效率为93%;敏感性高,约是常规PCR方法的100倍,最低可以检测到101拷贝/μL的模板;重复性好,批内和批间重复性试验变异系数分别为0.17%～0.90%和0.65%～2.34%;用本研究所建立的实时荧光定量检测方法对120份临床样品进行检测,PRRSV阳性检出率为59.2%(71/120),而常规PCR方法的PRRSV阳性检出率为44.2%(53/120)。该方法的建立为PRRSV的实验室诊断、流行病学调查,以及预防和控制中国PRRSV的流行提供了快速、准确的检测手段。
In order to quickly and accurately detect porcine reproductive and respiratory syndrome(PRRSV),one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain,highly pathogenic mutant strain,as well as the new NADC30-like strain at the same time.The specificity,sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Realtime RT-PCR method was specific for PRRSV classic strain,highly pathogenic mutant strain,aswell as the NADC30-like strain,and the test results were negative with CSFV,PEDV,TGEV,RV,PRV,PPV and PCV2.The method showed a good linear relationship within the template ranges from 101 to 108 copies/μL,and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999,and the efficiency was 93%.The limit of detection concentration was 101 copies/μL.The coefficient of variation in the intra-and inter-assays were0.17%to 0.90% and 0.65% to 2.34%,respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method(59.2%)was higher than that using the conventional PCR method(44.2%).The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.
In order to quickly and accurately detect porcine reproductive and respiratory syndrome(PRRSV),one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain,highly pathogenic mutant strain,as well as the new NADC30-like strain at the same time.The specificity,sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Realtime RT-PCR method was specific for PRRSV classic strain,highly pathogenic mutant strain,aswell as the NADC30-like strain,and the test results were negative with CSFV,PEDV,TGEV,RV,PRV,PPV and PCV2.The method showed a good linear relationship within the template ranges from 101 to 108 copies/μL,and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999,and the efficiency was 93%.The limit of detection concentration was 101 copies/μL.The coefficient of variation in the intra-and inter-assays were0.17%to 0.90% and 0.65% to 2.34%,respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method(59.2%)was higher than that using the conventional PCR method(44.2%).The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.
引文
[1]NEUMANN E J,KLIEBENSTEIN J B,JOHNSON C D,et al.Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States[J].Journal of the American Veterinary Medical Association,2005,227(3):385-392.
[2]NIEUWENHUIS N,DUINHOF T F,VANNES A.Economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds[J].Veterinary Record,2012,170(9):225-230.
[3]TIAN K,YU X,ZHAO T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One,2007,2(6):e526.
[4]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒TaqMan-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报,2008,30(12):944-948.WEI T C,TIAN Z J,AN T Q,et al.Development and application of TaqMan-MGB fluorescence quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(12):944-948.(in Chinese)
[5]LI Y,JI G,WANG J,et al.Complete genome sequence of an NADC30-like porcine reproductive and respiratory syndrome virus characterized by recombination with other strains[J].Genome Announcements,2016,4(3):e00330-16.
[6]王林建,郭振华,乔松林,等.河南地区NADC30-like PRRSV毒株的增殖特性与遗传进化分析[J].河南农业科学,2017,46(3):122-128.WANG L J,GUO Z H,QIAO S L,et al.Proliferation characteristics and phylogenetic analysis of one NADC30-like PRRSV strain isolated in Henan[J].Journal of Henan Agricultural Sciences,2017,46(3):122-128.(in Chinese)
[7]WANG L J,WAN B,GUO Z,et al.Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in China[J].Virus Genes,2018,54(1):86-97.
[8]ZHOU L,CHEN S,ZHANG J,et al.Molecular variation analysis of porcine reproductive and respiratory syndrome virus in China[J].Virus Resesrch,2009,145(1):97-105.
[9]TIAN K.NADC30-like porcine reproductive and respiratory syndrome in China[J].The Open Virology Journal,2017,30(11):59-65.
[10]周一媚,劳秀杰,黄巧莲,等.荧光定量RT-PCR检测PRRSV方法的建立及初步应用[J].畜牧与兽医,2016,48(1):34-39.ZHOU Y M,LAO X J,HUANG Q L,et al.Establishment and application of Real-time RT-PCR methods for detection of porcine reproductive and respiratory syndrome virus[J].Animal Husbandry&Veterinary Medicine,2016,48(1):34-39.(in Chinese)
[11]LIU J,ZHOU X,ZHAI J,et al.Recombination in JXA1-R vaccine and NADC30-like strain of porcine reproductive and respiratory syndrome viruses[J].Veterinary Microbiology,2017,204(5):110-120.
[12]WASILK A,CALLAHAN J D,CHRISTOPHER-HENNINGS J,et al.Detection of U.S.,Lelystad,and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by Real-time PCR[J].Journal of Clinical Microbiology,2004,42(10):4453-4461.
[13]CHUNG W B,CHAN W H,CHAUNG H C,et al.Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2in naturally-infected and challenged pigs[J].Journal of Virological Methods,2005,124(1-2):11-19.
[14]周璐,李淞,徐薇薇,等.猪环曲病毒RT-PCR检测方法的建立及其应用[J].中国兽医科学,2012,42(12):1264-1267.ZHOU L,LI S,XU W W,et al.Development and application of RT-PCR assay for detection of porcine torovirus[J].Chinese Veterinary Science,2012,42(12):1264-1267.(in Chinese)
[15]王淑娟,刘梅芬,闫若潜,等.猪瘟病毒实时荧光定量PCR检测方法的建立与应用[J].中国畜牧兽医,2017,44(7):2112-2118.WANG S J,LIU M F,YAN R Q,et al.Establishment and application of Real-time quantitative PCR assay for detection of classical swine fever virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):2112-2118.(in Chinese)
[16]万春和,刘荣昌,程龙飞,等.鸭腺病毒A型TaqMan实时荧光定量PCR检测方法的建立[J].中国畜牧兽医,2018,45(5):1341-1348.WAN C H,LIU R C,CHENG L F,et al.Establishment of a TaqMan-based Real-time PCR method of duck adenovirus A[J].China Animal Husbandry&Veterinary Medicine,2018,45(5):1341-1348.(in Chinese)
[17]郭广君,吕素芳,肖跃强,等.猪伪狂犬病病毒检测方法研究进展[J].动物医学进展,2011,32(12):84-88.GUO G J,LYU S F,XIAO Y Q,et al.Research progress on detection method of pseudorabies virus[J].Progress in Veterinary Medicine,2011,32(12):84-88.(in Chinese)
[18]李凯,高宏雷,高立,等.禽网状内皮组织增生病病毒TaqMan探针荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2010,40(11):1137-1141.LI K,GAO H L,GAO L,et al.Development and application of a TaqMan Real-time PCR assay for detection of reticuloendotheliosis virus[J].Chinese Veterinary Science,2010,40(11):1137-1141.(in Chinese)
[19]孔刚锐,吴志明,闫若潜,等.鉴别高、低致病性猪繁殖与呼吸道综合征病毒二重实时荧光定量RT-PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(12):27-33.KONG G R,WU Z M,YAN R Q,et al.Establisment and application of a double Real-time RT-PCR assay for the differential diagnosis of highly and lowly pathogenic porcine reproductive and respiratory syndrome virus[J].China Animal Husbandry&Veterinary Medicine,2013,40(12):27-33.(in Chinese)
[20]谢丽华,戴光文,植芝林.猪繁殖与呼吸综合征病毒RT-PCR鉴别诊断方法的建立及应用[J].畜牧与兽医,2016,48(12):99-102.XIE L H,DAI G W,ZHI Z L.Establishment and application of identifiable RT-PCR method for detecting porcine reproductive and respiratory syndrome virus[J].Animal Husbandry&Veterinary Medicine,2016,48(12):99-102.(in Chinese)
[21]张硕.猪繁殖与呼吸综合征病毒分离株遗传进化分析及通用实时荧光RT-PCR检测方法的建立和应用[D].北京:中国农业大学,2017.ZHANG S.Evolutionary analysis of porcine reproductive and respiratory syndrome virus and establishment of universal Real-time fluorescent RT-PCR detection method[D].Beijing:China Agricultural University,2017.(in Chinese)
[22]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,47(5):544-550.LI X J,GONG S Y,CHEN Y Q,et al.Establisment and application of a double RT-PCR for the detection of PRRSV and PEDV[J].Chinese Veterinary Science,2017,47(5):544-550.(in Chinese)
[23]李凯,高宏雷,高立,等.TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数[J].畜牧兽医学报,2011,42(5):742-746.LI K,GAO H L,GAO L,et al.Development of a Real-time PCR for determination of foreign gene copy number in genome of Pichia pastoris[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):742-746.(in Chinese)
[24]赵玲娜,金红岩,梁琳,等.实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立[J].中国畜牧兽医,2016,43(11):2844-2851.ZHAO L N,JIN H Y,LIANG L,et al.A SYBRGreenⅠbased on Real-time quantitative RT-PCR assay for specific detection of peste des petits rumanants virus[J].China Animal Husbandry&Veterinary Medicine,2016,43(11):2844-2851.(in Chinese)
[2]NIEUWENHUIS N,DUINHOF T F,VANNES A.Economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds[J].Veterinary Record,2012,170(9):225-230.
[3]TIAN K,YU X,ZHAO T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One,2007,2(6):e526.
[4]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒TaqMan-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报,2008,30(12):944-948.WEI T C,TIAN Z J,AN T Q,et al.Development and application of TaqMan-MGB fluorescence quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(12):944-948.(in Chinese)
[5]LI Y,JI G,WANG J,et al.Complete genome sequence of an NADC30-like porcine reproductive and respiratory syndrome virus characterized by recombination with other strains[J].Genome Announcements,2016,4(3):e00330-16.
[6]王林建,郭振华,乔松林,等.河南地区NADC30-like PRRSV毒株的增殖特性与遗传进化分析[J].河南农业科学,2017,46(3):122-128.WANG L J,GUO Z H,QIAO S L,et al.Proliferation characteristics and phylogenetic analysis of one NADC30-like PRRSV strain isolated in Henan[J].Journal of Henan Agricultural Sciences,2017,46(3):122-128.(in Chinese)
[7]WANG L J,WAN B,GUO Z,et al.Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in China[J].Virus Genes,2018,54(1):86-97.
[8]ZHOU L,CHEN S,ZHANG J,et al.Molecular variation analysis of porcine reproductive and respiratory syndrome virus in China[J].Virus Resesrch,2009,145(1):97-105.
[9]TIAN K.NADC30-like porcine reproductive and respiratory syndrome in China[J].The Open Virology Journal,2017,30(11):59-65.
[10]周一媚,劳秀杰,黄巧莲,等.荧光定量RT-PCR检测PRRSV方法的建立及初步应用[J].畜牧与兽医,2016,48(1):34-39.ZHOU Y M,LAO X J,HUANG Q L,et al.Establishment and application of Real-time RT-PCR methods for detection of porcine reproductive and respiratory syndrome virus[J].Animal Husbandry&Veterinary Medicine,2016,48(1):34-39.(in Chinese)
[11]LIU J,ZHOU X,ZHAI J,et al.Recombination in JXA1-R vaccine and NADC30-like strain of porcine reproductive and respiratory syndrome viruses[J].Veterinary Microbiology,2017,204(5):110-120.
[12]WASILK A,CALLAHAN J D,CHRISTOPHER-HENNINGS J,et al.Detection of U.S.,Lelystad,and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by Real-time PCR[J].Journal of Clinical Microbiology,2004,42(10):4453-4461.
[13]CHUNG W B,CHAN W H,CHAUNG H C,et al.Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2in naturally-infected and challenged pigs[J].Journal of Virological Methods,2005,124(1-2):11-19.
[14]周璐,李淞,徐薇薇,等.猪环曲病毒RT-PCR检测方法的建立及其应用[J].中国兽医科学,2012,42(12):1264-1267.ZHOU L,LI S,XU W W,et al.Development and application of RT-PCR assay for detection of porcine torovirus[J].Chinese Veterinary Science,2012,42(12):1264-1267.(in Chinese)
[15]王淑娟,刘梅芬,闫若潜,等.猪瘟病毒实时荧光定量PCR检测方法的建立与应用[J].中国畜牧兽医,2017,44(7):2112-2118.WANG S J,LIU M F,YAN R Q,et al.Establishment and application of Real-time quantitative PCR assay for detection of classical swine fever virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):2112-2118.(in Chinese)
[16]万春和,刘荣昌,程龙飞,等.鸭腺病毒A型TaqMan实时荧光定量PCR检测方法的建立[J].中国畜牧兽医,2018,45(5):1341-1348.WAN C H,LIU R C,CHENG L F,et al.Establishment of a TaqMan-based Real-time PCR method of duck adenovirus A[J].China Animal Husbandry&Veterinary Medicine,2018,45(5):1341-1348.(in Chinese)
[17]郭广君,吕素芳,肖跃强,等.猪伪狂犬病病毒检测方法研究进展[J].动物医学进展,2011,32(12):84-88.GUO G J,LYU S F,XIAO Y Q,et al.Research progress on detection method of pseudorabies virus[J].Progress in Veterinary Medicine,2011,32(12):84-88.(in Chinese)
[18]李凯,高宏雷,高立,等.禽网状内皮组织增生病病毒TaqMan探针荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2010,40(11):1137-1141.LI K,GAO H L,GAO L,et al.Development and application of a TaqMan Real-time PCR assay for detection of reticuloendotheliosis virus[J].Chinese Veterinary Science,2010,40(11):1137-1141.(in Chinese)
[19]孔刚锐,吴志明,闫若潜,等.鉴别高、低致病性猪繁殖与呼吸道综合征病毒二重实时荧光定量RT-PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(12):27-33.KONG G R,WU Z M,YAN R Q,et al.Establisment and application of a double Real-time RT-PCR assay for the differential diagnosis of highly and lowly pathogenic porcine reproductive and respiratory syndrome virus[J].China Animal Husbandry&Veterinary Medicine,2013,40(12):27-33.(in Chinese)
[20]谢丽华,戴光文,植芝林.猪繁殖与呼吸综合征病毒RT-PCR鉴别诊断方法的建立及应用[J].畜牧与兽医,2016,48(12):99-102.XIE L H,DAI G W,ZHI Z L.Establishment and application of identifiable RT-PCR method for detecting porcine reproductive and respiratory syndrome virus[J].Animal Husbandry&Veterinary Medicine,2016,48(12):99-102.(in Chinese)
[21]张硕.猪繁殖与呼吸综合征病毒分离株遗传进化分析及通用实时荧光RT-PCR检测方法的建立和应用[D].北京:中国农业大学,2017.ZHANG S.Evolutionary analysis of porcine reproductive and respiratory syndrome virus and establishment of universal Real-time fluorescent RT-PCR detection method[D].Beijing:China Agricultural University,2017.(in Chinese)
[22]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,47(5):544-550.LI X J,GONG S Y,CHEN Y Q,et al.Establisment and application of a double RT-PCR for the detection of PRRSV and PEDV[J].Chinese Veterinary Science,2017,47(5):544-550.(in Chinese)
[23]李凯,高宏雷,高立,等.TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数[J].畜牧兽医学报,2011,42(5):742-746.LI K,GAO H L,GAO L,et al.Development of a Real-time PCR for determination of foreign gene copy number in genome of Pichia pastoris[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):742-746.(in Chinese)
[24]赵玲娜,金红岩,梁琳,等.实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立[J].中国畜牧兽医,2016,43(11):2844-2851.ZHAO L N,JIN H Y,LIANG L,et al.A SYBRGreenⅠbased on Real-time quantitative RT-PCR assay for specific detection of peste des petits rumanants virus[J].China Animal Husbandry&Veterinary Medicine,2016,43(11):2844-2851.(in Chinese)