口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用

doi:10.16431/j.cnki.1671-7236.2017.02.002

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (2): 311-318.doi: 10.16431/j.cnki.1671-7236.2017.02.002

口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用

姚怀兵^1,2, 赵毅³, 刘宏³, 张毅³, 任方³, 黄炯²

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

收稿日期:2016-07-21 出版日期:2017-02-20 发布日期:2017-02-25
通讯作者: 黄炯 E-mail:jh124@163.com
作者简介:姚怀兵(1990-), 男, 新疆五家渠人, 硕士生, 研究方向:预防兽医学, E-mail:389691747@qq.com
基金资助:
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）

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

YAO Huai-bing^1,2, ZHAO Yi³, LIU Hong³, ZHANG Yi³, REN Fang³, HUANG Jiong²

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

Received:2016-07-21 Online:2017-02-20 Published:2017-02-25

摘要/Abstract

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

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.

Key words: type O foot and mouth disease virus; two-temperature RT-PCR; detection method

中图分类号:

S855.3

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.

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]. , 2017, 44(2): 311-318.

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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

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

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