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口蹄疫病毒可视化RT-LAMP检测方法的建立
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引用本文:王韦华,刘桂梅,吴奇强,黄鹏波,白 鸽,王国超.口蹄疫病毒可视化RT-LAMP检测方法的建立[J].西北农业学报,2018,27(12):1736~1744
DOI:10.7606/j.issn.1004-1389.2018.12.005
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王韦华,刘桂梅,吴奇强,黄鹏波,白 鸽,王国超 (1.渭南职业技术学院,陕西渭南 714000 2.渭南市动物疫病预防控制中心,陕西渭南 714000) 
基金项目:陕西省农业科技创新转化项目(NYKJ-2015-021)。
中文摘要:旨在建立口蹄疫病毒可视化逆转录环介导等温核酸扩增(RT-LAMP)快速检测方法。以猪口蹄疫灭活疫苗为材料,设计两对RT-LAMP引物,建立FMDV可视化RT-LAMP检测方法,并与普通PCR检测方法进行比较,同时进行特异性试验和敏感性试验。对从油佐剂疫苗中提取FMDV核酸方法进行比较结果发现,用商品化试剂盒提取时,3种样品处理方法对病毒RNA提取量没有显著影响;Trizol法提取RNA时,从油佐剂疫苗中直接提取,获得的RNA量最大,与其他2种方法差异极显著(P<0.01)。建立了口蹄疫病毒可视化RT-LAMP方法,该方法检测到模板RNA最低稀释度为10-9,比RT-PCR方法灵敏10倍,特异性好。
中文关键词:口蹄疫病毒  逆转录环介导等温核酸扩增  检测
 
Establishment of a Visible Detection of Foot-and-Mouth Disease Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay
Abstract:To establish a visible detection of foot-and-mouth disease virus(FMDV) by Reverse Transcription Loop-Mediated Isothermal Amplification(RT-LAMP),using the vaccine against foot and mouth disease as materials and designing two RT-LAMP primers,the FMDV visualization RT-LAMP detection method was established ,and compared with the common PCR method.The specificity test and sensitivity test were carried out simultaneously.The results showed that based on the comparison of the extraction of FMDV nucleic acid from oil adjuvant vaccine,the three sample treatments had no significant effect on the RNA extraction of the virus when extracted with the commercialization kit.The trizol method was used to extract RNA,and the amount of RNA obtained by direct extraction of oil adjuvant vaccine was the highest,which was significantly different from that of the other two methods(P<0.01).A visualization RT-LAMP method to detection foot-and-mouth disease virus was established.The method was high specificity with the lowest detection limit of 10-9 dilution of RNA sample,10-fold higher than that of RT-PCR,showed the method.The novel assay RT-LAMP was a rapid,simple,sensitive,and specific test for FMDV,and it could be applied in field conditions.
keywords:Foot-and-mouth disease virus(FMDV)  Reverse transcription loop-mediated isothermal amplification(RT-LAMP)  Detection
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