猪瘟病毒基因分型寡核苷酸芯片方法的建立

根据猪瘟病毒（CSFV）E2基因序列,设计41条针对CSFV 3个基因群共10个亚群各亚群寡核苷酸探针。利用欧盟猪瘟诊断手册推荐的CSFV E2基因套式RT-PCR方法,在内套PCR过程中进行Cy3-dCTP掺入荧光标记,制备芯片杂交样品。用标记的PCR产物与寡核苷酸探针阵列杂交,置于GenePix 4100A扫描仪中扫描,利用Ge-nePix Pro 6.0软件分析杂交图像。特异性和灵敏度试验显示,芯片方法与本室发表的CSFV real-time RT-PCR方法的灵敏度相近,芯片探针与猪繁殖与呼吸综合征病毒（PRRSV）、猪2型圆环病毒（PCV2）、猪伪狂犬病毒（PRV）样品无非特异性杂交。以包括1.1、2.1、2.2、2.3亚群的8份CSF阳性样品进行芯片的检测验证,结果表明,通过特异性的杂交图谱或杂交信号分析可准确判定样品所属的基因亚群,寡核苷酸芯片的检测结果与测序的分型结果全部符合。本研究为将寡核苷酸芯片技术用于猪瘟病毒的基因分型和分子流行病学研究奠定了基础。

Establishment of oligonucleotide microarray assay for genotyping of CSFV

Corresponding to the sequence of CSFV E2 gene,41 specific oligonucleotide probes were designed and were used to manufacture the microarray to differentiate 10 subgroups of 3 gene groups of CSFV.The CSFV E2 gene specific nested RT-PCR method recommended by the EU diagnosis manual for CSF was deployed to amplify and label the targets,Cy3-dCTP was incorporated in the inner set PCR.The labelled PCR products were hybridized with the oligonucleotide microarray,the microarray image was captured with GenePix 4100A scanner and analysed with a GenePix Pro 6.0 software.The sensitivity of this assay was equal to the published CSFV real-time RT-PCR method developed by our research group,the oligonucleotide probes did not show cross reaction with PRRSV,PCV2 and PRV nucleic acids.The microarray was applied to test 8 CSF positive samples representing subgroup1.1,subgroup2.1,subgroup2.2 and subgroup2.3.The results showed that genotype of the samples could be determined by hybridization pattern or the analysis of the fluorescent signals.The results of the microarray were the same with that obtained by sequencing.The present study may provide a basis for the further application of oligonucleotide microarray for genotyping of CSFV and molecular epidemiology.