猪圆环病毒3型PCR检测方法的建立及其应用  

DEVELOPMENT AND APPLICATION OF PCR METHOD FOR DETECTION OF PORCINE CIRCOVIRUS TYPE 3

作　　者：肖琦[1] 茅爱华[1] 汪伟[1] 俞正玉[1] 郭佳慧 袁万哲[3] 赵攀登 温立斌[1] 范宝超[1] 李彬[1] 朱雪蛟 胡屹屹[1] 郭容利[1] 曲梦 杨倩[6] 何孔旺[1] XIAO Qi;MAO Ai-hua;WANG Wei;YU Zheng-yu;GUO Jia-hui;YUAN Wan-zhe;ZHAO Pan-deng;WEN Li-bin;FAN Bao-chao;LI Bin;ZHU Xue-jiao;HU Yi-yi;GUO Rong-li;QU Meng;YANG Qian;HE Kong-wang(Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture.Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;College of Veterinary Medicine,Agricultural University of Hebei,Baoding 071001,China;College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Agriculture and Animal Husbandry,Tibet University,Linzhi 860000,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室,南京210014 [2]扬州大学兽医学院,扬州225009 [3]河北农业大学动物医学院,保定071001 [4]河南牧业经济学院动物医学院,郑州450046 [5]西藏大学农牧学院,林芝860000 [6]南京农业大学动物医学院,南京210095

出　　处：《中国动物传染病学报》2018年第2期9-14,共6页Chinese Journal of Animal Infectious Diseases

基　　金：中国农业科学院基本科研业务费专项院级统筹工作项目(Y2017PT50)

摘　　要：根据Gen Bank登录的猪圆环病毒3型(porcine circovirus type 3,PCV3)全基因组序列设计并合成了一对特异性引物,经条件优化,建立了检测PCV3的PCR方法,扩增片段大小为932 bp;最低的质粒检测浓度为10 copy/μL;对猪圆环病毒1型、猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪细小病毒的核酸进行扩增,均无PCV3目的条带出现;随机抽取扩增的16份临床病猪样品目的条带进行TA克隆后测序,结果显示均为PCV3特异性目的片段,16株PCV3江苏毒株扩增序列与已报道的PCV3/US/SD2016和PCV3/CN/Hubei-618基因序列同源性分别为98.4%~99.6%、99.0%~99.8%,而16个毒株之间的同源性为98.3%~100%。利用所建立的PCR方法检测了2014~2017年间收集的江苏省临床病猪样品938份、健康猪样品500份,PCV3的阳性检出率分别为6.93%和0.6%;其中,2014年病猪样品中的阳性率为1.92%,后呈逐渐上升的趋势,2017年达到12.67%。本研究表明,建立的PCR方法特异、敏感,可以用于临床PCV3的检测;PCV3在江苏省猪群2014年已有感染,并呈逐年升高的趋势,应予以重视。To detect porcine circovirus type 3(PCV3),an emerging porcine infectious agent,from clinical samples quickly and specifi cally,a pair of specifi c primers was designed and synthesized based on the complete genome sequence of PCV3 of GenBank to develop a PCR method.The amplifi ed fragment was 932 bp in length.The classic plasmid concentration with PCR detection was 10 copy/μL for PCV3 but no amplifi cation from nucleic acids of PCV1,PCV2,swine fever virus,Porcine reproductive and respiratory syndrome virus,Pseudorabies virus and Porcine parvovirus.The amplifi ed fragments from 16 clinical samples of pigs were cloned and sequenced.The results showed that the nucleotide sequences shared 98.4%-99.6%and 99.0%-99.8%identity with the reported PCV3 SD2016 strain in the United States and Hubei-618 strain in China.The nucleotide homology between the obtained 16 strains was 98.3%-100%.Then,the PCR method was used to test 938 clinical samples collected from 2014 to 2017 in diseased pigs and 500 healthy pig samples in Jiangsu province.As a result,the positive rates of PCV3 were 6.93%and 0.6%,respectively.In addition,the positive rate was 1.92%in 2014 and then gradually increased to 12.67%in 2017.This study showed that the established PCR method is specifi c and sensitive and might be used for the detection of clinical PCV3 samples.

关 键 词：圆环病毒3型 PCR 临床应用 

分 类 号：S852.659.2[农业科学—基础兽医学]