CSFV、PRV和PRRSV多重PCR检测方法的建立及其应用
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摘要
由猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)引起的繁殖障碍性疫病在中国的不断暴发,增加了猪的发病率和死亡率。为了建立同时检测这3种病毒的多重PCR方法,本研究根据GenBank中这3种病毒的参考基因序列设计引物,并对反应中的影响因素进行优化,建立了同时检测CSFV、PRV和PRRSV的多重PCR方法。该方法扩增的基因片段大小分别为570(CSFV),232 (PRRSV)和173bp(PRV)。敏感性和特异性试验结果显示,该方法对3种病毒的核酸最低检出量分别为23.88(CSFV),13.10(PRRSV)和14.60pg(PRV),对大肠杆菌(Ec.oli)、沙门菌(Salmonella)、猪乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的检测结果均为阴性。对2015年5月至2016年1月收集的168份临床样本检测结果显示,CSFV阳性率为24.4%,PRRSV阳性率为21.4%,提示河北省该段时间内引起猪繁殖障碍性疫病的主要病原为CSFV和PRRSV。经多重PCR检测为PRRSV、PRV或CSFV阳性的临床样本,再次使用商品化的试剂盒进行检测,符合率为100.0%。这些结果表明,本试验建立的多重PCR方法省时、高效,可用于PRRSV、PRV和CSFV感染的临床诊断。
Outbreaks of reproductive failure disease in swine caused by classical swine fever virus(CSFV),porcine pseudorabies virus(PRV)and porcine reproductive and respiratory syndrome virus(PRRSV)have increased the morbidity and mortality of pig in China.We developed a novel multiplex assay based on reverse transcription and polymerase chain reaction(PCR)for the simultaneous detection of CSFV,PRV and PRRSV.The multiplex PCR assay produced 570,232 and173 bp amplicons for CSFV,PRRSV and PRV,respectively.The sensitivity of the multiplex PCR assay was evaluated using extracted nucleic acids containing the target sequences,and the detection limits were 23.88,13.10,and 14.60 pg for CSFV,PRRSV and PRV,respectively.A total of 168 clinical samples which were collected from aborted and dead fetuses during May 2015 to January2016 were detected using the multiplex PCR assay.The CSFV-positive rate was 24.4% and PRRSV-positive rates was 21.4%,suggesting that CSFV and PRRSV were the major causes of swine reproductive disease in Hebei province during that period.The positive samples of PRRSV,PRV or CSFV were re-detected by commercial Kits.The detect results were the same as that of the multiplex PCR.The developed multiplex PCR assay is an efficient and time saving method,and can be used for the clinical diagnosis of PRRSV,PRV and CSFV infection.
Outbreaks of reproductive failure disease in swine caused by classical swine fever virus(CSFV),porcine pseudorabies virus(PRV)and porcine reproductive and respiratory syndrome virus(PRRSV)have increased the morbidity and mortality of pig in China.We developed a novel multiplex assay based on reverse transcription and polymerase chain reaction(PCR)for the simultaneous detection of CSFV,PRV and PRRSV.The multiplex PCR assay produced 570,232 and173 bp amplicons for CSFV,PRRSV and PRV,respectively.The sensitivity of the multiplex PCR assay was evaluated using extracted nucleic acids containing the target sequences,and the detection limits were 23.88,13.10,and 14.60 pg for CSFV,PRRSV and PRV,respectively.A total of 168 clinical samples which were collected from aborted and dead fetuses during May 2015 to January2016 were detected using the multiplex PCR assay.The CSFV-positive rate was 24.4% and PRRSV-positive rates was 21.4%,suggesting that CSFV and PRRSV were the major causes of swine reproductive disease in Hebei province during that period.The positive samples of PRRSV,PRV or CSFV were re-detected by commercial Kits.The detect results were the same as that of the multiplex PCR.The developed multiplex PCR assay is an efficient and time saving method,and can be used for the clinical diagnosis of PRRSV,PRV and CSFV infection.
引文
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[12]罗尚星,范京惠,刘宝京,等.副猪嗜血杆菌、PCV2及与PRRSV多联RT-PCR检测方法的建立及其应用[J].中国兽医学报,2018,38(8):1513-1518.
[13]陈钟鸣,张定东,方光远,等.PRRSV、CSFV和JEV混合感染的多联PCR诊断[J].动物医学进展,2008,29(12):23-27.
[14]王娟萍,姚敬明,高英杰,等.多重PCR/RT-PCR技术检测PRRSV、PPV、PRV和PCV-2[J].中国兽医学报,2009,29(3):258-262.
[15]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.
[16]孙文超,刘立明,赵翠青,等.猪细小病毒6型SYBRGreenⅠreal-time PCR检测方法的建立[J].中国兽医学报,2018,38(3):442-445,452.
[17]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.
[18]徐朋丽,郑慧华,赵宇,等.猪圆环病毒2型SYBR-GreenⅠ实时荧光定量PCR检测方法的建立[J].中国兽医学报,2018,38(9):1655-1660.
[2]CHEN H Y,WEI Z Y,ZHANG H Y,et al.Use of a multiplex RT-PCR assay for simultaneous detection of the North American genotype porcine reproductive and respiratory syndrome virus,swine influenza virus and Japanese encephalitis virus[J].Sci Agri Sin,2010,9(7):1050-1057.
[3]XU X G,CHEN G D,HUANG Y,et al.Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses[J].J Virol Methods,2012,183(1):69-74.
[4]ZENG Z,LIU Z,WANG W,et al.Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine[J].J Virol Methods,2014,208:102-106.
[5]李斌,马俊杰,毛立,等.猪Hokovirus PCR检测方法的建立及其应用[J].中国预防兽医学报,2012,34(2):108-111.
[6]LIU J K,WEI C H,YANG X Y,et al.Multiplex PCRfor the simultaneous detection of porcine reproductive and respiratory syndrome virus,classical swine fever virus,and porcine circovirus in pigs[J].Mol Cell Probes,2013,27(3/4):149-152.
[7]WU H,RAO P,JIANG Y,et al.A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders[J].Mol Cell Probes,2014,28(5/6):264-270.
[8]JIANG Y,SHANG H,XU H,et al.Simultaneous detection of porcine circovirus type 2,classical swine fever virus,porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction[J].Vet J,2010,183(2):172-175.
[9]LIU S S,ZHAO Y R,HU Q B,et al.A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus,classical swine fever virus,and porcine reproductive and respiratory syndrome virus in clinical specimens[J].J Virol Methods,2011,172(1/2):88-92.
[10]HRI CˇíNOVáM,HOLODA E,MUDROňOVáD,et al.Multiplex PCR assay for detection of Actinobacillus pleuropneumoniae,Pasteurella multocida and Haemophilus parasuis in lungs of pigs from a slaughterhouse[J].Folia Microbiol,2010,55(6):635-640.
[11]KANG I,KIM D,HAN K,et al.Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis,Streptococcus suis,and Mycoplasma hyorhinis in formalinfixed,paraffin-embedded tissues from pigs with polyserositis[J].Canadian J Vet Res,2012,76(3):195-200.
[12]罗尚星,范京惠,刘宝京,等.副猪嗜血杆菌、PCV2及与PRRSV多联RT-PCR检测方法的建立及其应用[J].中国兽医学报,2018,38(8):1513-1518.
[13]陈钟鸣,张定东,方光远,等.PRRSV、CSFV和JEV混合感染的多联PCR诊断[J].动物医学进展,2008,29(12):23-27.
[14]王娟萍,姚敬明,高英杰,等.多重PCR/RT-PCR技术检测PRRSV、PPV、PRV和PCV-2[J].中国兽医学报,2009,29(3):258-262.
[15]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.
[16]孙文超,刘立明,赵翠青,等.猪细小病毒6型SYBRGreenⅠreal-time PCR检测方法的建立[J].中国兽医学报,2018,38(3):442-445,452.
[17]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.
[18]徐朋丽,郑慧华,赵宇,等.猪圆环病毒2型SYBR-GreenⅠ实时荧光定量PCR检测方法的建立[J].中国兽医学报,2018,38(9):1655-1660.