猪流行性腹泻病毒和猪圆环病毒3型二重SYBR GreenⅠ实时荧光定量PCR方法的建立
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摘要
为建立同时快速检测猪流行性腹泻病毒(PEDV)和猪圆环病毒3型(PCV3)的二重PCR方法,本研究根据GenBank中登录的PEDV M基因和PCV3 Rep基因高度同源保守序列设计并合成2对特异性引物,分别扩增PEDV M基因与PCV3 Rep基因,经过反应条件优化,建立了检测PEDV与PCV3的二重SYBR GreenⅠ实时荧光定量PCR方法。结果显示:该方法对PEDV和PCV3的检测结果为阳性,而对猪圆环病毒2型、猪传染性胃肠炎病毒和猪轮状病毒等8种常见的病原体均未检测到荧光信号;且具有良好的线性关系,R~2为0.99;应用本方法对PEDV和PCV3的最低检出量分别为37.6拷贝/μL和61.2拷贝/μL;组内和组间变异系数均小于2%。结果表明:本试验建立的PEDV和PCV3二重实时荧光定量PCR方法具有良好的特异性、敏感性、重复性,为快速、高效、定量检测PEDV和PCV3提供了技术帮助。
To develop a rapid duplex PCR assay for simultaneous detection of porcine epidemic diarrhea virus(PEDV) and porcine circovirus type 3(PCV3).In this study,two pairs of primers were designed and synthesized according to the conserved regions of the M gene of PEDV and the Rep gene of PCV3 published in GenBank,and a SYBR Green Ⅰ-based qPCR assay for detection of PEDV and PCV3 was developed by optimizing the amplification conditions.The results demonstrated that the assay was specific to detect PEDV and PCV3,whereas no fluorescence signal was detected for the common pathogens such as porcine circovirus type 2,porcine transmissible gastroenteritis virus,porcine rotavirus,porcine bocavirus,pseudorabies virus,porcine parvovirus,swine fever virus,porcine reproductive and respiratory syndrome virus,etc.The assay showed a good linear relationship,and the minimum detectable amount was 37.6 copies/μL for PEDV and 61.2 copies/μL for PCV3;the co-efficient of variations in intra-and inter-assay were both less than 2%.The results showed that the method has good specificity,sensitivity and repeatability,which provides technical support for the rapid,efficient and quantitative detecting the clinical samples with PEDV and PCV3 or co-infections.
To develop a rapid duplex PCR assay for simultaneous detection of porcine epidemic diarrhea virus(PEDV) and porcine circovirus type 3(PCV3).In this study,two pairs of primers were designed and synthesized according to the conserved regions of the M gene of PEDV and the Rep gene of PCV3 published in GenBank,and a SYBR Green Ⅰ-based qPCR assay for detection of PEDV and PCV3 was developed by optimizing the amplification conditions.The results demonstrated that the assay was specific to detect PEDV and PCV3,whereas no fluorescence signal was detected for the common pathogens such as porcine circovirus type 2,porcine transmissible gastroenteritis virus,porcine rotavirus,porcine bocavirus,pseudorabies virus,porcine parvovirus,swine fever virus,porcine reproductive and respiratory syndrome virus,etc.The assay showed a good linear relationship,and the minimum detectable amount was 37.6 copies/μL for PEDV and 61.2 copies/μL for PCV3;the co-efficient of variations in intra-and inter-assay were both less than 2%.The results showed that the method has good specificity,sensitivity and repeatability,which provides technical support for the rapid,efficient and quantitative detecting the clinical samples with PEDV and PCV3 or co-infections.
引文
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[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 Green Ⅰ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] 徐丽丽,邓慧华,赵宇,等.猪圆环病毒3型SYBR Green Ⅰ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.
[19] WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus Res,2016,226:7-13.
[20] LI W,LI H,LIU Y,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerg Infect Dis,2012,18(8):1350-1353.
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[22] SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerg Infect Dis,2012,18(1):161-163.
[23] 陈建飞,刘孝珍,时洪艳,等.2010-2011年仔猪腹泻的病因分析和防控措施[J].养猪,2011(5):81-83.
[24] CHEN G H,TANG X Y,SUN Y,et al.Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs[J].J Virol Methods,2018,251:129-132.
[25] MILLER L C,CRAWFORD K K,LAGER K M,et al.Evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs[J].J Vet Diagn Invest,2016,28(1):20-29.
[2] SUN M,MA J,WANG Y,et al.Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia[J].J Clin Microbiol,2015,53(5):1484-1492.
[3] HORIE M,KABEMURA M,MASATANI T,et al.Isolation and molecular characterization of porcine epidemic diarrhea viruses collected in Japan in 2014[J].Arch Virol,2016,161(8):2189-2195.
[4] CHUNG H C,NGUYEN V G,MOON H J,et al.Isolation of porcine epidemic diarrhea virus during outbreaks in South Korea,2013-2014[J].Emerg Infect Dis,2015,21(12):2238-2240.
[5] PALINSKI R,PI?EYRO P,SHANG P,et al.A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol,2016,91(1):e01879-16.
[6] KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[7] STADEJEK T,WO■,et al.First detection of porcine circovirus type 3 on commercial pig farms in Poland[J].Transbound Emerg Dis,2017,64(5):1350-1353.
[8] KWON T,YOO S J,PARK C K,et al.Prevalence of novel porcine circovirus 3 in Korean pig populations[J].Vet Microbiol,2017,207:178-180.
[9] FACCINI S,BARBIERI I,GILIOLI A,et al.Detection and genetic characterization of porcine circovirus type 3 in Italy[J].Transbound Emerg Dis,2017,64(6):1661-1664.
[10] PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2016,13:184.
[11] WANG J,ZHANG Y,ZHANG R,et al.Recombinase polymerase amplification assay for rapid detection of porcine circovirus 3[J].Mol Cell Probes,2017,36:58-61.
[12] ZHAI S L,ZHOU X,ZHANG H,et al.Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations[J].Virol J,2017,14(1):222.
[13] 贺会利,李军,潘艳,等.广西首例猪圆环病毒3型的发现及其衣壳蛋白序列分析[J].南方农业学报,2017,48(8):1499-1503.
[14] 张贺,李波,周虚,等.实时荧光定量PCR技术研究进展及应用[J].动物医学进展,2006,27(1):5-12.
[15] 修金生,周伦江,陈如敬,等.猪流行性腹泻病毒SYBRI实时荧光定量RT-PCR检测方法的建立[J].中国兽医科学,2012(2):160-165.
[16] 刘邓,袁秀芳,冉多良,等.TaqMan荧光定量PCR检测猪流行性腹泻病毒方法的建立与初步应用[J].中国动物传染病学报,2010,18(1):28-34.
[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 Green Ⅰ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] 徐丽丽,邓慧华,赵宇,等.猪圆环病毒3型SYBR Green Ⅰ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.
[19] WANG D,FANG L,XIAO S.Porcine epidemic diarrhea in China[J].Virus Res,2016,226:7-13.
[20] LI W,LI H,LIU Y,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerg Infect Dis,2012,18(8):1350-1353.
[21] CHEN J,LIU X,SHI D,et al.Complete genome sequence of a porcine epidemic siarrhea virus variant[J].J Virol,2012,86(6):3408.
[22] SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerg Infect Dis,2012,18(1):161-163.
[23] 陈建飞,刘孝珍,时洪艳,等.2010-2011年仔猪腹泻的病因分析和防控措施[J].养猪,2011(5):81-83.
[24] CHEN G H,TANG X Y,SUN Y,et al.Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs[J].J Virol Methods,2018,251:129-132.
[25] MILLER L C,CRAWFORD K K,LAGER K M,et al.Evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs[J].J Vet Diagn Invest,2016,28(1):20-29.