猪病毒性繁殖障碍病低密度基因芯片诊断方法的建立

Low Density DNA Array for Diagnosing Swine Viral Reproductive Disease

应用PCR(或RT-PCR)分别扩增猪繁殖与呼吸障碍综合征病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒-2型、日本乙型脑炎病毒和猪瘟病毒的一段保守序列,克隆到pMD 18-T Simple Vector构建重组质粒,用于制备各病毒的探针。将各探针按一定的阵列点加到硝酸纤维素膜上并加以固定,制备成低密度检测基因芯片。在多重PCR扩增过程中用生物素标记的dUTP(Bio-11-dUTP)对待检样品进行标记,扩增产物与芯片杂交,用扫描仪对杂交结果进行扫描分析。经对68份样品检测结果证明,该方法可以同时检测待检样品中上述6种病毒核酸的存在情况,与PCR方法相比,显示了较高的灵敏度。该方法的建立,为大批量快速诊断、普查猪病毒性繁殖障碍疫病提供了保证。

Conserved DNA（or cDNA）fragments of six viruses （porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine cirocovirus-2, porcine parvovirus, porcine psudorabies virus and Japanese B encephalitis virus）, obtained by using PCR （or RT-PCR）, were ligated with pMD 18-T Simple Vector to construct recombinant plasmid for DNA probe preparation. Then the purified DNA probes were spotted on located sites on nitrocellulose to prepare low density DNA array. During the process of multiple PCR, specimens were labeled by Biotin-ll-dUTP. And the labeled PCR products were hybridized with DNA array, the results were scanned and analyzed by scanning device and software. The results showed that this DNA array could identify and distinguish the six viruses mentioned above in specimen at same time and was more sensitive than PCR method. This method can be use for lab diagnosis, large scale epidemiology investigation and quarantine of animal and animal products.