【摘要】 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。更多还原

【Abstract】 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-stranded-DNA targets and the targets were labeled with Cy-dye.Following purification,the labeled asymmetrical PCR products were hybridized to the microarrays.The presence of the virulence genes was determined by a fluorescent signal above the background noise.When the spots of capturing probes is corresponded with samples a positive result displayed,while in the negative and blank controls showed no signal.In conclusion,for its high sensitivity,good specificity,simplicity,the technique can be used for diagnosis and prevention of the porcine viral(PPV,PCV-2,CSFV)infections.更多还原