口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用

试验通过对口蹄疫病毒核苷酸序列的比对分析，在O型口蹄疫病毒的P1基因保守区，设计1对特异性引物，应用均匀设计法优化反应参数，建立口蹄疫O型病毒二温式RT-PCR检测方法。对该法进行特异性试验、敏感性试验检测。结果表明，该二温式RT-PCR方法只对口蹄疫O型病毒敏感，对其他血清型的口蹄疫病毒及常见的猪病病毒均不敏感；扩增条带与预期目的片段大小相符，扩增片段经克隆、测序发现与引物所在基因序列的同源性为100%；检测病毒RNA的敏感性为1.665 pg/μL，其敏感性与三步法PCR敏感性检测结果没有差异。运用该法对54头攻毒试验的动物进行检测，阳性鉴定结果与三步法PCR鉴定结果一致，与三步法PCR相比该法节省了20 min，表明所建立的口蹄疫O型病毒二温式RT-PCR方法是一种准确、快速、特异、敏感的检测方法。

Development and Application of Two-temperature RT-PCR for Detectionof Type O Foot and Mouth Disease Virus

According to the gene sequences analysis of foot and mouth disease virus (FMDV) in GenBank,a pair of specific primers was designed in the conserved sequence of type O FMDV P1 gene. The reaction parameters were optimized using the uniform design method to develop a two-temperature RT-PCR method for detection of type O FMDV.The results of sensitivity and specificity showed that the two-temperature RT-PCR method was only specific for type O FMDV without amplification of the other viruses. The amplified fragment was same with the expected length.The cloning and sequencing results revealed that the sequence of amplified fragment had 100% simililarity to the target sequence,and the minimum detection quantity was 1.665 pg/μL,the effective detection rate was consistent with the three step RT-PCR sensitivity test results. 54 taper toxicity test pigs were detected,and positive identification results and three-step PCR results was consistent.Compared with the three-step PCR,it could save 20 min.These results indicated that the developed two-temperature RT-PCR for detection of type O FMDV was a kind of accurate,rapid,specific and sensitive detection method.