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| 口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用 | |
| 引用本文： | 姚怀兵,赵毅,刘宏,张毅,任方,黄炯.口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用[J].中国畜牧兽医,2017,44(2):311-318. |
| 作者姓名： | 姚怀兵 赵毅 刘宏 张毅 任方 黄炯 |
| 作者单位： | 1. 新疆农业大学动物医学学院, 乌鲁木齐 830052; 2. 新疆畜牧科学院兽医研究所, 乌鲁木齐 830000; 3. 天康生物股份有限公司, 乌鲁木齐 830000 |
| 基金项目： | 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） |
| 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1.665 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。 |
| 关 键 词： | 口蹄疫O型病毒 二温式RT-PCR 检测方法 |
| 收稿时间： | 2016-07-21 |
Development and Application of Two-temperature RT-PCR for Detectionof Type O Foot and Mouth Disease Virus |
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| YAO Huai-bing,ZHAO Yi,LIU Hong,ZHANG Yi,REN Fang,HUANG Jiong.Development and Application of Two-temperature RT-PCR for Detectionof Type O Foot and Mouth Disease Virus[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):311-318. | |
| Authors: | YAO Huai-bing ZHAO Yi LIU Hong ZHANG Yi REN Fang HUANG Jiong |
| Institution: | 1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China; 2. Institute of Veterinary Medicine, Xinjiang Academy of Animal Sciences, Urumqi 830000, China; 3. Tecon Bio-technology Co., Urumqi 830000, China |
| Abstract: | 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. |
| Keywords: | type O foot and mouth disease virus two-temperature RT-PCR detection method |
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