猪圆环病毒2型单管巢式PCR检测方法建立  

Establishment of Single Tube Nested PCR for Detection of Porcine Circovirus Type 2

作　　者：夏嘉鑫 岳丰雄 林艳 XIA Jiaxin;YUE Fengxiong;LIN Yan(Chengdu Tianbang Biological Products Co.,Ltd.,Chengdu 610100,China)

机构地区：[1]成都天邦生物制品有限公司,四川成都610100

出　　处：《养猪》2022年第1期95-100,共6页Swine Production

摘　　要：猪圆环病毒2型(Porcine circovirus 2,PCV2)是引起断奶仔猪多系统衰竭综合征的主要病原体。为建立一种灵敏度更高且特异性更强的分子检测技术,研究建立一种猪圆环病毒2型单管巢式PCR检测技术。首先利用梯度PCR方法验证内外引物的最佳退火温度;再利用正交试验设计的方法对单管巢式PCR反应体系的内外引物浓度、Ex Taq酶用量等相关因素进行优化。结果表明,当外引物PCV2-P1/P3退火温度为64.4℃,内引物PCV2-1/2退火温度为54.4℃,通过正交试验最终筛选出的最佳反应体系(25μL)为:2×Taq PCR Mastermix 10μL,100μmol/L的内引物PCV2-1/2各1μL,10μmol/L的外引物PCV2-P1/P3各0.2μL,模板DNA 2.5μL,ddH_(2)O 10.1μL。上述反应体系仅对猪圆环病毒2型扩增出特异性目的条带,具有高度特异性,最低检测下限针对猪圆环病毒2型DBN-SX07株目的基因的最低DNA模板量为0.6×10^(-6) ng、ZJ/c株目的基因的最低DNA模板量为0.6×10^(-3) ng。研究建立的猪圆环病毒2型单管巢式PCR检测方法可以有效针对猪圆环病毒2型特异性检测。Porcine circovirus 2(PCV2)is the main pathogen causing multisystem failure syndrome in weaned piglets.In order to establish a more sensitive and specific molecular detection technology,a single tube nested PCR detection technology of porcine circovirus type 2 was established.Firstly,gradient PCR was used to verify the optimal annealing temperature of internal and external primers;the results showed that when the annealing temperature of outer primer PCV2-P1/P3 was 64.4℃and the annealing temperature of inner primer PCV2-1/2 was 54.4℃,the best reaction system was finally selected by orthogonal test(25μL)is:2×Taq PCR Mastermix 10μL,100μmol/L inner primer PCV2-1/2 each 1μL,10μmol/L external primer PCV2-P1/P3 each 0.2μL.Template DNA 2.5μL,ddH_(2)O 10.1μL.The above reaction system only amplified specific bands for porcine circovirus type 2,with high specificity.The minimum detection limit for the target gene of porcine circovirus type 2 DBN-SX07 strain was 0.6×10^(-6) ng.The minimum amount of DNA template of target gene of 10^(-6) ng and ZJ/c strains was 0.6×10^(-3) ng.The detection method of porcine circovirus type 2 by single tube nested PCR established in this study can effectively detect porcine circovirus type 2.

关 键 词：猪圆环病毒2型 单管巢式PCR DBN-SX07株 ZJ/c株 

分 类 号：S858.285.3[农业科学—临床兽医学]