猪圆环病毒3型与猪圆环病毒2型二重PCR检测方法的建立
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摘要
为建立同时检测PCV3和PCV2的二重PCR方法,分别以PCV3和PCV2基因组的保守区域设计2对特异性引物,片段大小分别为649,295 bp。将反应条件进行优化,构建PCV3和PCV2的二重PCR检测方法。检测结果表明该方法能特异性扩增PCV3和PCV2,且无法扩增其他猪常见病毒;PCV3和PCV2的最低检出限分别为508,412 copies/μL。临床样品检测结果显示,PCV3和PCV2阳性率分别为2.1%(3/145),62.1%(90/145),PCR产物经测序证实为PCV3。本研究建立的二重PCR方法具有速度快、特异性强和灵敏度等优点,可以用于PCV3和PCV2的检测和流行病学调查。
To develop a duplex PCR assay for the detection PCV3 and PCV2,two pairs of specific primers were designed according to the genome conservative sequence of PCV3 and PCV2,which one was used for specifically amplification of a 645 bp fragment of PCV3,and the other for amplification of a 295 bp fragment of PCV2.The reaction conditions of the annealing temperature and the primer concentration were optimized.Specificity test showed no amplification was observed when other common pathogens for canine gastrointestinal diseases were used as template.The detection limits of assay were 508 copies/μL for PCV3 DNA and 412 copies/μL for PCV2 DNA.Furthermore,the established assay was used to detect clinical samples collected from Guangxi.The results showed that the positive rates of PCV3 and PCV2 were2.1%(3/145)and 62.1%(90/145),respectively.These results suggested that the developed duplex PCR assay was suitable for clinical detection and epidemiology surveillance of PCV3 and PCV2.
To develop a duplex PCR assay for the detection PCV3 and PCV2,two pairs of specific primers were designed according to the genome conservative sequence of PCV3 and PCV2,which one was used for specifically amplification of a 645 bp fragment of PCV3,and the other for amplification of a 295 bp fragment of PCV2.The reaction conditions of the annealing temperature and the primer concentration were optimized.Specificity test showed no amplification was observed when other common pathogens for canine gastrointestinal diseases were used as template.The detection limits of assay were 508 copies/μL for PCV3 DNA and 412 copies/μL for PCV2 DNA.Furthermore,the established assay was used to detect clinical samples collected from Guangxi.The results showed that the positive rates of PCV3 and PCV2 were2.1%(3/145)and 62.1%(90/145),respectively.These results suggested that the developed duplex PCR assay was suitable for clinical detection and epidemiology surveillance of PCV3 and PCV2.
引文
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[2] NOVOSEL D,TUBOLY T,CSAGOLA A,et al.Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia[J].Vet Rec,2014,174(17):431.
[3] PARK C,SEO H W,PARK S J,et al.Comparison of porcine circovirus type 2 (PCV2)- associated lesions produced by co-infection between two genotypes of PCV2 and two genotypes of porcine reproductive and respiratory syndrome virus[J].J Gen Virol,2014,95(11):2486-2494.
[4] SHIN M K,YOON S H,KIM M H,et al.Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems[J].J Vet Sci,2015,16(1):25-29.
[5] JEONG J,PARK C,CHOI K,et al.Comparison of three commercial one-dose porcine circovirus type 2 (PCV2) vaccines in a herd with concurrent circulation of PCV2b and mutant PCV2b[J].Vet Microbiol,2015,177(1-2):43-52.
[6] ZHANG D,HE K,WEN L,et al.Genetic and phylogenetic analysis of a new porcine circovirus type 2 (PCV2) strain in China[J].Arch Virol,2015,160(12):3149-3151.
[7] PHAN TG,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(1):184.
[8] FAN S,KU X,CHEN F,et al.Complete Genome Sequence of a Novel Porcine Circovirus Type 3 Strain,PCV3/CN/Hubei-618/2016,Isolated from China[J].Genome Announc,2017,5(15).
[9] PHAN TG,GIANNITTI F,ROSSOW S,et al.Erratum to:Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2017,14(1):87.
[10] JIANG Y,FANG L,SHI X,et al.Simultaneous detection of five enteric viruses associated with gastroenteritis by use of a PCR assay:a single real-time multiplex reaction and its clinical application[J].J Clin Microbiol,2014,52(4):1266-1268.
[11] FAN W L,WANG Z W,QIN Y,et al.Use of dual priming oligonucleotide system-based multiplex RT-PCR combined with high performance liquid chromatography assay for simultaneous detection of five enteric viruses associated with acute enteritis[J].J Virol Methods,2017,243:80-82.
[12] AFGHAH Z,WEBB B,MENG X J,et al.Ten years of PCV2 vaccines and vaccination:Is eradication a possibility?[J].Vet Microbiol,2017,206:21-28.
[13] TUMMARUK P,PEARODWONG P.Expression of PCV2 antigen in the ovarian tissues of gilts[J].J Vet Med Sci,2016,78(3):457-461.
[14] ZHANG G,JIA P,CHENG G,et al.Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides[J].Carbohydr Polym,2017,166:64-72.
[15] KAIDA A,KUBO H,SEKIGUCHI J,et al.Detection of five rash-associated viruses using multiplex real-time PCR during 2006-2011[J].Jpn J Infect Dis,2012,65(5):430-432.
[16] HORWOOD P F,MAHONY T J.Multiplex real-time RT-PCR detection of three viruses associated with the bovine respiratory disease complex[J].J Virol Methods,2011,171(2):360-363.