PRV和PCV2二重PCR检测方法的建立与初步应用
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摘要
为建立同时检测猪伪狂犬病病毒(pseudorabies virus,PRV)和猪圆环病毒2型(porcine circovirus 2,PCV2)2种病毒的多重PCR,设计2对PRV和PCV2特异性引物,建立同时检测这2种病毒的二重PCR方法,并对采自呼吸障碍病猪的病料进行检测。结果表明,PRV和PCV2扩增产物分别为359bp和482bp,最小DNA质量分别是2.7pg和4.3pg。采自河南省的80份样品的总阳性率为85%(68/80),其中PRV和PCV2阳性率分别为28.8%(23/80)和77.5%(62/80),混合感染率达25%(17/68)。该多重PCR检测方法敏感、特异、快速,可用于临床样品PRV和PCV2检测。
To simultaneously detect pseudorabies virus(PRV)and porcine circovirus type 2(PCV2),two pairs of primers specific to PRV and PCV2 are designed and a dulplex PCR was developed.The samples from pigs with respiratory failure were detected by the dulplex PCR.The results showed that products for PRV and PCV2 are 359bp and 482 bp,respectively.The DNA detection limit of the multiplex PCR was 2.7pg and 4.3pg for PRV and PCV2,respectively.Among 100 pig specimens from Henan province,the total positive rate was 85%(68/80)and within them,the positive rate for PRV and PCV2 was respectively 28.8%(23/80)and 77.5%(62/80).And the co-infection rate was25%(17/68).This dulplex PCR is sensitive,specific and rapid method for the detection and identification of PRV and PCV2 in clinical samples.
To simultaneously detect pseudorabies virus(PRV)and porcine circovirus type 2(PCV2),two pairs of primers specific to PRV and PCV2 are designed and a dulplex PCR was developed.The samples from pigs with respiratory failure were detected by the dulplex PCR.The results showed that products for PRV and PCV2 are 359bp and 482 bp,respectively.The DNA detection limit of the multiplex PCR was 2.7pg and 4.3pg for PRV and PCV2,respectively.Among 100 pig specimens from Henan province,the total positive rate was 85%(68/80)and within them,the positive rate for PRV and PCV2 was respectively 28.8%(23/80)and 77.5%(62/80).And the co-infection rate was25%(17/68).This dulplex PCR is sensitive,specific and rapid method for the detection and identification of PRV and PCV2 in clinical samples.
引文
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[3]SHU Xianghua(舒相华),YIN Gefen(尹革芬),YANG Zhilei(杨志雷),et al.An Epidemiological Survey on PCV-2,CSFV,PRRSV and PRV that Play as Main Pathogens of PRDC in Some Herds of Swine in Yunnan Areas[J].Journal of Yunnan Agricultural University(云南农业大学学报),2011,26(1):54-58(in Chinese with English abstract).
[4]Zhao Z,Qin Y,Lai Z,et al.Microbial ecology of swine farms and PRRS vaccine vaccination strategies[J],2012,155(2-4):247-56.
[5]Edwards M C,Gibbs R A.Multiplex PCR:advantages,development,and applications[J].PCR Methods Applications,1994,3(4):S65-S75.
[6]WANG Longbo(王隆柏),ZHANG Zhigang(张志刚),SU Yongyu(苏永裕),et al.Establishment of the multiplex PCR for detection of 5kinds of swine disease[J].China Animal Husbandry&Veterinary Medicine(中国畜牧兽医),2014,41(2):21-25(in Chinese with English abstract).
[7]Xie Z,Luo S,Xie L,et al.Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-basedmultiplex PCR assay[J].Journal of Virological Methods,2014,207:188-195.
[8]Pang Y,Wang H,Girshick T,et al.Development and application of a multiplex polymerase chain reaction for avian respiratory agents[J].Avian Diseases,2002,46(3):691-699.
[9]Gutierrez-Martin C B,Rodriguez-Delgado O,Alvarez-Nistal D,et al.Simultaneous serological evidence of Actinobacillus pleuropneumoniae,PRRS,Aujeszky’s disease and influenza viruses in Spanish finishing pigs[J].Research Veterinary Science,2000,68(1):9-13.
[10]Vlasakova M,Leskova V,Sliz I,et al.The presence of six potentially pathogenic viruses in pigs suffering from postweaning multisystemic wasting syndrome[J].BMC Veterinary Research,2014,10:221.
[11]Hansen M S,Pors S E,Jensen H E,et al.An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark[J].Journal of Comparative Pathology,2010,143(2-3):120-131.
[12]Opriessnig T,Giménez-Lirola L G,Halbur P G.Polymicrobial respiratory disease in pigs[J].Animal Health Research Reviews,2011,2(2):133-148.
[13]Fablet C,Marois-Créhan C,Simon G,et al.Infectious agents associated with respiratory diseases in 125farrow to finish pig herds:a cross-sectional study[J].Veterinary Microbiology,2012,157(1-2):152-163.
[14]Grau-Roma L,Segalés J.Detection of porcine reproductive and respiratory syndrome virus,porcine circovirus type 2,swine influenza virus and Aujeszky’s disease virus in cases of porcine proliferative and necrotizing pneumonia(PNP)in Spain[J].Veterinary Microbiology,2007,19(2-4):144-151.
[15]Bochev I.Porcine respiratory disease complex(PRDC):a review.Ⅰ.Etiology,epidemiology,clinical forms and pathoanatomical features[J].Bulgarian Journal of Veterinary Medicine,2007,10(3):131-146.