猪3种重要病毒寡核苷酸芯片诊断方法的建立

Establishment of oligonucleotide microarray diagnostic technique for porcine three viral infections

将猪细小病毒(PPV)、猪圆环病毒-Ⅱ(PCV-2)、猪瘟病毒(CSFV)分别应用生物信息学方法,针对病毒基因组保守序列设计特异性强的60-mer寡核苷酸探针,并将其按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片。分别设计出相应的引物,对待测样本进行不对称PCR扩增,从而产生大量可与寡核苷酸探针特异性互补的单链DNA片段,并通过间接荧光标记技术使扩增产物标记上荧光染料。将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交,扫描、分析芯片上荧光信号。试验结果表明,芯片上各样本对应探针位点呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。不对称PCR技术制备的单链DNA片段与寡核苷酸芯片进行杂交反应可同时、快速、特异性地检测多种猪疫病病毒。

The oligonucleotide microarray coupled with asymmetrical PCR system was developed to detect simultaneously,rapidly and specifically the multiple viral infections of swine snchas porcine parvovirus,porcine circovirus and classical swine fever virus.Species-specific 60-mer oligonucleotide probes(oligoprobes) acted as the capture probes were designed and immobilized on the surface of amino modified slides.Asymmetrical PCR was used to prepare abundant single-stranded target DNA.The aa-dUTP was mixed into single...