口蹄疫病毒分型诊断芯片探针设计初报

Preliminary Study on Microarray Probe Designing for FMDV Genotyping

为建立口蹄疫病毒(Foot-and-mouth disease virus,FMDV)不同血清型与基因型的基因芯片检测方法,设计针对O型8个基因型、A型3个基因型和亚洲1型的特异性探针。从美国GenBank与英国世界口蹄疫参考实验室基因库下载了O型、A型和亚洲1型FMDV的VP1基因序列547条。对每一血清型序列用DNA Star软件ClastalW程序进行多重比对,做系统发育分析并进行基因分型。用生物学软件BioSun 2.0建立基因型数据库,设计每一基因型的特异性探针。共设计出104条候选探针,通过芯片试验筛选出12条特异性探针。以各型特异性探针所对应的靶序列模板做10倍系列稀释进行PCR扩增,扩增产物与探针杂交,验证各探针的灵敏度。对O型SEA、Euro-SA、ME-SA、WA 4个基因型的各条探针的灵敏度进行了检验,结果这些探针能够检测到102数量级拷贝数的阳性靶标。

To develop a microarray for FMDV genotyping and serotyping, genotype specific oligonucleotide probes for eight genotypes in serotype O FMDV, three genotypes in serotype A and Asia I FMDV were generated. A total of 547 complete VP1 gene sequences of serotype O, A and Asia I were downloaded from NCBI and WRLFMD GenBanks. The genotypes of the sequences were determined by Megalignment comparison and phylogenetic analysis of the VP1 gene using DNA Star ClustalW program and TreeView program. BioSun 2. 0 software was used for genotype based database setting and oligonucleotide probe designing within each targeted genotype. A total of 104 candidate probes were designed and 12 of them were confirmed to be genotype specific by microarray testing. The PCR amplified target samples from cloned templates and their 10 time serial dilutions of the target samples were hybridized with the candidate probes to determine their sensitivity. The sensitivity test with 1 probe in each of 4 genotypes in type O FMDV showed that the probes were able to detect minimum 10^2 copies of positive targets.