中国动物检疫2016，Vol.33Issue(12)：78-84,7.DOI:10.3969/j.issn.1005-944X.2016.12.022
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
摘要
Abstract
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Key words
xMAP array/porcine reproductive failure virus/detection methods分类
农业科技引用本文复制引用
杨显超,吴秀娟,李凯航,杨德全,鞠厚斌,葛菲菲,王建..液相芯片技术同时检测六种猪繁殖障碍病毒的方法研究[J].中国动物检疫,2016,33(12):78-84,7.基金项目
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 （）
