液相芯片技术同时检测六种猪繁殖障碍病毒的方法研究  

Establishment of a Multiplex Supension Array for Simultaneous Detection of Six Kinds of Porcine Reproductive Failure Virus

作　　者：杨显超[1] 吴秀娟[1] 李凯航[1] 杨德全[1] 鞠厚斌[1] 葛菲菲[1] 王建[1] 

机构地区：[1]上海市动物疫病预防控制中心,上海201103

出　　处：《中国动物检疫》2016年第12期78-84,共7页China Animal Health Inspection

基　　金：上海市市级农口系统青年人才成长计划(沪农青字〔2014〕第2-11号);上海市生猪产业技术体系(沪农科产字〔2016〕第6号)

摘　　要：在GenBank中收集猪细小病毒（PPV）结构蛋白VP2基因、猪圆环病毒2型（PCV2）的ORF2序列、猪伪狂犬病毒（PRV）的gB基因、猪繁殖与呼吸综合征病毒（PRRSV）的NSP2基因、猪瘟病毒（CSFV）的E2基因和乙型脑炎病毒（JEV）的M基因，利用Primer 5.0等分子生物学软件对收集的序列分析比较，筛选出上述基因的特异保守序列并设计引物和探针，将设计好的探针与相应的微球偶联，优化条件，建立检测方法。结果表明：建立的检测PPV、PCV2、PRV、PRRSV、CSFV、JEV 6种病毒核酸的多重液相芯片技术具有较好的特异性、敏感性和稳定性，对PPV、PCV2、PRV、PRRSV、CSFV、JEV 6种病毒核酸的最低检出量分别为8.50×102 copies/μL、2.87×102 copies/μL、2.07×102 copies/μL、2.61×102 copies/μL、2.34×102 copies/μL、2.05×102 copies/μL，与相应病毒PCR/RT-PCR检测方法相比，敏感性提高了100-1000倍；对临床388份样品同步进行荧光PCR与液相芯片检测，结果99.87%相符。本方法为液相芯片技术在动物多病毒快速高通量检测、鉴别诊断等应用方面奠定了基础。From more than 20 articles published in GenBank,structural protein VP2 gene sequence of porcine parvovirus（PPV）,open reading frame 2 sequence of porcine circovirus 2（PCV2）,gB genome sequence of pseudorabies virus（PRV）,NSP2 genome sequence of swine reproductive and respiratory syndrome virus（PRRSV）, E2 genome sequence of classical swine fever virus（CSFV）and M genome sequence of Japanese encephalitis virus（JEV）, were downloaded and multi-sequence analysis for filtering out the conserved region by DNAman V6 were done. Then the sequences were inputted into Primer 5.0 software to design out combinations of the specific probes and primers of PPV,PCV2,PRV,PRRSV,CSFV and JEV. Next,the probes and microspheres were coupled,reverse primers and probes’reverse complementary sequence were tagged with biotin. Through the optimization of the annealing hybridization temperature and other reaction conditions,the xMAP instrument system was established for detection of PPV,PCV2,PRV,PRRSV,CSFV and JEV. The results showed that the specificity of combinations of each pair primers and probes was good,and there was no cross-reaction with each other. This method also could be sensitive to detect the six viruses,the limit of detection for each nucleic acid were 8.50×102copies/μL,2.87×102 copies/μL, 2.07×102 copies/μL,2.61×102 copies/μL,2.34×102 copies/μL,2.05×102 copies/μL,respectively,its sensitivity increased 100~1 000 times compared with PCR/RT-PCR of each corresponding virus. 388 clinical samples were tested by the xMAP methods and ordinary PCR/RT-PCR methods respectively at same time,and the results showed that consistent test results were obtained by this two methods. The xMAP methods laid foundation for application of xMAP technology in fast,high-throughput detection of multi-virus of animals and differential diagnosis.

关 键 词：液相芯片技术 猪繁殖障碍病毒 检测方法 

分 类 号：S851.3[农业科学—预防兽医学]