猪六种重要病原寡核苷酸芯片检测方法的初步建立  

Development of oligonucleotide microarray for simultaneous detection of six swine pahtogens

作　　者：保雨 聂福平[2,3] 杨俊[2,3] 王昱[2,3] 刘亚娟[1,2] 王国民[2] 李贤良[2] 李应国[2] 张超[2] 刘力[1] 

机构地区：[1]西南大学动物科技学院,重庆400715 [2]重庆出入境检验检疫局/重庆市进出口食品安全工程中心,重庆400020 [3]重庆大学生物工程学院,重庆400030

出　　处：《中国兽医科学》2016年第2期142-148,共7页Chinese Veterinary Science

基　　金：质检公益性项目(201310093);国家质检总局科技项目(2014IK247;2015IK332)

摘　　要：为建立同时鉴别检测猪传染性胸膜肺炎放线杆菌（APP）、副猪嗜血杆菌（HPS）、猪繁殖与呼吸综合征病毒（PRRSV）、猪肺炎支原体（Mhp）、猪圆环病毒2型（PCV2）和猪瘟病毒（CSFV）这6种病原的寡核苷酸芯片方法,根据这6种病原的特异保守序列,设计了6对特异性引物和探针,建立多重PCR体系,优化杂交参数,进行方法评估。结果显示,该方法检测APP、HPS、Mhp、PCV2、PRRSV及CSFV的灵敏度分别为5.8×10^2copies/L、7.8×10^3copies/L、6.8×10^3copies/L、6.3×10^2copies/L、4.8×10^3copies/L、5.5×10^2copies/L,且与猪源伪狂犬病病毒、猪细小病毒、日本乙型脑炎病毒、猪水疱病病毒、水泡性口炎病毒、口蹄疫病毒、蓝舌病病毒、小反刍兽疫病毒及沙门菌等9种病原无交叉反应。用建立的多重PCR对186份临床样本的检测结果显示,单一感染率为18.3%（34/186）,混合感染率为5.9%（11/186）,与测序验证完全一致。该方法为多种病原的流行病学调查和临床诊断提供了一种快速、高通量的检测手段。In order to establish an oligonucleotide microarray for simultaneous detection of Actinobacillus pleuropneumoniae（APP）,Haemophilus parasuis（HPS）,porcine reproductive and respiratory syndrome virus（PRRSV）, Mycoplasma hyopneumoniae（Mhp）, porcine circovirus type 2（PCV2）,and classical swine fever virus（CSFV）, six pairs of primers and probes were designed based on specific and conservative sequences of six pathogens. Under the optimum condition,the detection limits of oligonucleotide microarray were up to 5.8×10^2copies/ L for APP, 7.8×10^3copies/ L for HPS, 6.8×10^3copies/ L for Mhp, 6.3×10^2copies/ L for PCV2, 4.8×10^3copies/ L for PRRSV and 5.5×10^2copies/ L for CSFV, and had no cross-reaction with pseudorabies virus,porcine parvovirus, Japanese B encephalitis virus, swine vesicular disease virus, vesicular stomatitis virus, foot-and-mouth disease virus, bluetongue virus,peste des petits ruminants virus and Salmonella,respectively. Moreover, to evaluate the potential application of the oligonucleotide microarray in clinical diagnosis, 186 clinical samples from pig were tested. The positive rates of single infection and co-infection were 18.3% and 5.9%, respectively,the results were consistent with DNA sequencing. Therefore, the established method provides a rapid and high-throughput method for the epidemiological investigation and clinical diagnosis of six pathogens.

关 键 词：寡核苷酸芯片 APP HPS MHP PCV2 PRRSV CSFV 

分 类 号：S858.28[农业科学—临床兽医学]