摘要
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1.665pg/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20min,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。
According to the gene sequences analysis of foot and mouth disease virus（FMDV）in GenBank,apair 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 twotemperature 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 had100% simililarity to the target sequence,and the minimum detection quantity was 1.665pg/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.
出处
《中国畜牧兽医》
CAS
北大核心
2017年第2期311-318,共8页
China Animal Husbandry & Veterinary Medicine
基金
自治区科研机构创新发展专项资金(2016D04008)
自治区产学研联合培养研究生示范基地项目(xjaucxy-yjs-20152008)
关键词
口蹄疫O型病毒
二温式RT-PCR
检测方法
type O foot and mouth disease virus
two-temperature RT-PCR
detection method
