猪圆环病毒2型和3型双重PCR检测方法的建立
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摘要
猪圆环病毒3型(PCV3)是一种新的猪圆环病毒,与猪皮炎和肾病综合征、母猪繁殖障碍及心脏和多系统炎症相关。为了建立一种同时快速检测猪圆环病毒2型(PCV2)和PCV3的双重PCR方法,根据GenBank收录的PCV2和PCV3基因序列,分别在PCV2 Cap基因和PCV3 Rep基因高度同源保守区设计并筛选2对特异性引物,经过双重PCR反应条件的优化,建立了同时检测PCV2/3的双重PCR检测方法。本方法可同时扩增出PCV2、PCV3的486,270bp特异性片段,而扩增猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)等病原核酸结果均为阴性;PCV2/3最低检出量分别为139,21.7拷贝/μL。经临床应用表明,本试验建立的双重PCR方法简便、快速,敏感度高,特异性强,为PCV2/3的鉴别诊断和联合检测提供了依据。
Porcine circovirus type 3(PCV3)has been recently proposed as a new porcine circovirus.It is associated with porcine dermatitis and nephropathy syndrome,reproductive failure and systemic inflammation disease.To develop a rapid duplex PCR assay for simultaneous detection of PCV2 and PCV3,two pairs of specific primers were designed and synthesized according to the published complete genome sequences of PCV2 and PCV3in GenBank.A duplex PCR method was developed by optimizing the amplification conditions to simultaneously detect PCV2 and PCV3.The results showed that two specific fragments of 486 bp for PCV2 and 270bp for PCV3 could simultaneously be amplified in the duplex PCR,whereas no PCR products were amplified from nucleic acid from porcine reproductive and respiratory syndrome virus,classical swine fever virus,pseudorabies virus,porcine epidemic diarrhea virus,porcine parvovirus,swine salmonellosis,Escherichia coli,Haemophilus parasuis,respectively.The detection limits of the method were 139copies/μL for PCV2 and 21.7copies/μL for PCV3,respectively.Clinical detection of PCV2/3by the duplex PCR method showed that the duplex PCR method established in this study has simple and fast,high sensitivity and good specificity,and provides support for differentiation of PCV2/3and mixed infection.
Porcine circovirus type 3(PCV3)has been recently proposed as a new porcine circovirus.It is associated with porcine dermatitis and nephropathy syndrome,reproductive failure and systemic inflammation disease.To develop a rapid duplex PCR assay for simultaneous detection of PCV2 and PCV3,two pairs of specific primers were designed and synthesized according to the published complete genome sequences of PCV2 and PCV3in GenBank.A duplex PCR method was developed by optimizing the amplification conditions to simultaneously detect PCV2 and PCV3.The results showed that two specific fragments of 486 bp for PCV2 and 270bp for PCV3 could simultaneously be amplified in the duplex PCR,whereas no PCR products were amplified from nucleic acid from porcine reproductive and respiratory syndrome virus,classical swine fever virus,pseudorabies virus,porcine epidemic diarrhea virus,porcine parvovirus,swine salmonellosis,Escherichia coli,Haemophilus parasuis,respectively.The detection limits of the method were 139copies/μL for PCV2 and 21.7copies/μL for PCV3,respectively.Clinical detection of PCV2/3by the duplex PCR method showed that the duplex PCR method established in this study has simple and fast,high sensitivity and good specificity,and provides support for differentiation of PCV2/3and mixed infection.
引文
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[2]CAI L,HAN X,HU D,et al.A Novel Porcinecircovirus type 2astrain,10JS-2,with eleven-nucleotide insertions in the origin of genome replication[J].J Virol,2012,86(12):7017.
[3]江戈龙,高文超,刘德武,等.母源抗体对感染PCV2仔猪体内病毒变化和免疫反应的影响[J].中国兽医学报,2017,37(3):398-403.
[4]PALINSKI R,PINEYRO P,SHANG P,et al.A novel porcinecircovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol,2016,91(1):pii:e01879-16.
[5]PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novelcircovirus PCV3in pigs with cardiac and multisystemic inflammation[J].Virol J,2016,13(1):184.
[6]KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcinecircovirus type 3in China[J].Transbound Emerg Dis,2017,64(3):1865-1674.
[7]WANG J,ZHANG Y,WANG J,et al.Development of aTaqMan-based real-time PCR assay for the specific detection of porcinecircovirus 3[J].J Virol Methods,2017,248:177-180.
[8]CHEN G H,MAI K J,ZHOU L,et al.Detection and genome sequencing of porcinecircovirus 3in neonatal pigs with congenital tremors in South China[J].Transbound Emerg Dis,2017,64(6):1650-1654.
[9]KIM H R,PARK Y R,LIM D R,et al.Multiplex realtime polymerase chain reaction for the differential detection of porcinecircovirus 2and 3[J].J Virol Methods,2017,250:11-16.
[10]湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.
[11]郑玉姝,赵朴,刘俊伟,等.猪圆环病毒2型PCR检测方法的建立及应用[J].中围畜牧兽医,2010,37(8):66-68.
[12]徐朋丽,张鸿鑫,赵宇,等.猪圆环病毒3型PCR方法的建立与应用[J].中国预防兽医学报,2017,39(9):727-730.
[13] ZHENG S,WU X,ZHANG L,et al.The occurrence of porcinecircovirus 3without clinical infection signs in Shandong province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[14] STADEJEK T,WONIAK A,MIEK D,et al.First detection of porcine circovirus type 3on commercial pig farms in Poland[J].Transbound Emerg Dis,2017,64(5):1350-1353.
[15] FACCINI S,BARBIERI I,GILIOLI A,et al.Detection and genetic characterization of porcinecircovirus type3in Italy[J].Transbound Emerg Dis,2017,64(6):1661-1664.
[16] CHEN G H,TANG X Y,SUN Y,et al.Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3in pigs[J].J Virol Metheds,2017,251:129-132.
[17]付朋飞,乔涵,潘鑫龙,等.猪博卡病毒和猪圆环病毒2型双重PCR检测方法的建立[J].中国预防兽医学报,2014,36(9):708-711.
[18]周忠涛,王小敏,汪伟,等.2种猪圆环病毒2型TaqMan实时荧光定量PCR检测方法的建立及比较[J].农业科学与技术,2015,16(1):3-8.