三种鱼类弹状病毒实时荧光RT-LAMP检测试剂盒的建立与初步应用
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- 英文论文题名：Creation and Application of fish rhabdovirus real-time fluorescent RT-LAMP assay kit
- 论文作者：陈进会 ; 陈珍容 ; 陈洵 ; 唐大运 ; 颜其贵
- 英文论文作者：Chen Jinhui ; Chen Zhenrong ; Chen Xun ; Tang Dayun ; Yan Qigui ; Dongguan Entry-Exit Inspection and Quarantine Bureau ; Veterinary Medicine College of Sichuan Agricultural University ; Guangzhou Di Ao biotechnology limited company
- 年：2016
- 作者机构：东莞出入境检验检疫局;四川农业大学动物医学院;广州迪澳生物科技有限公司;
- 论文关键词：鱼类弹状病毒 ; 恒温实时荧光 ; 环介导等温扩增 ; 检测试剂盒
- 英文论文关键词：Rhabdovirus ; Isothermal realtime fluorescence assay ; LAMP ; Detection kit
- 会议召开时间：2016-06-14
- 会议录名称：第25届广东省科技进步活动月畜牧兽医学术与科技创新发展大会论文集
- 语种：中文
- 分类号：S941.41
- 学会代码：GDKL
- 会议名称：第25届广东省科技进步活动月畜牧兽医学术与科技创新发展大会
- 会议地点：中国广东广州
- 主办单位：广东省畜牧兽医学会
- 学会名称：广东省科学技术协会科技交流部
- 页数：10
- 文件大小：1626k
- 原文格式：O
- 会议级别：地方
摘要
构建检测三种鱼类弹状病毒实时荧光环介导等温RNA扩增(LAMP)试剂盒,用于现代口岸快速检测。对LAMP试剂进行组合优化,采用荧光染料显色方法判定LAMP结果,避免污染,减少假阳性结果产生;将以上优化技术及试剂整合成时荧光LAMP试剂盒;将建立的LAMP检测试剂盒和FQ-PCR检测试剂盒对样品进行对比试验。结果表明,研制的FQ-LAMP试剂盒与市售FQ-PCR试剂盒敏感性无差异,两者符合率100%,检出低限分别为2.4pg、3.6pg和4.5Pg;检测病毒IPNV、HRV和PFRV,无假阳性或假阴性,特异性强;构建的阳性质粒20次冻融和-20℃半年保存试验,稳定性好。三种弹状病毒FQ-LAMP试剂盒检测斑马鱼样品,证实三者引物无交叉反应,与FQ-PCR试剂盒检测结果一致。该试剂盒具有操作简单,便携,成本低,灵敏度高、特异性强,实验过程的数据化和自动化,适合现场检测和大规模监控。
To Construct a real-time fluorescence loop-mediated isothermal RNA amplification(LAMP) kit to rapidly detect three kinds of fish rhabdovirus,this kit will be used to quickly check of the modern port,LAMP reagents for combinatorial optimization,chromogenic method using fluorescent dye determination LAMP results,avoid contamination,reducing false positive result;the more optimization techniques and integrated into a fluorescent reagent kit LAMP;the establishment of LAMP assay kit and FQ- PCR detection kit samples were compared.The results showed that the sensitivity developed FQ-LAMP-free kit with commercial FQ-PCR kit difference between 100 percent compliance rate,low detection limits were 2.4pg,3.6pg and 4.5pg;detect viruses IPNV,HRV and PFRV,no false positives or false negatives,specificity;positive plasmid of 20 months-20 °C freezing and storage test,good stability.Three rhabdovirus FQ-LAMP kit to detect zebrafish samples confirmed the three primers no cross-reactivity,consistent with the FQ-PCR kit test results.The kit is simple,portable low cost,high sensitivity and specificity,the experimental data and automated processes for large-scale field testing and monitoring.
To Construct a real-time fluorescence loop-mediated isothermal RNA amplification(LAMP) kit to rapidly detect three kinds of fish rhabdovirus,this kit will be used to quickly check of the modern port,LAMP reagents for combinatorial optimization,chromogenic method using fluorescent dye determination LAMP results,avoid contamination,reducing false positive result;the more optimization techniques and integrated into a fluorescent reagent kit LAMP;the establishment of LAMP assay kit and FQ- PCR detection kit samples were compared.The results showed that the sensitivity developed FQ-LAMP-free kit with commercial FQ-PCR kit difference between 100 percent compliance rate,low detection limits were 2.4pg,3.6pg and 4.5pg;detect viruses IPNV,HRV and PFRV,no false positives or false negatives,specificity;positive plasmid of 20 months-20 °C freezing and storage test,good stability.Three rhabdovirus FQ-LAMP kit to detect zebrafish samples confirmed the three primers no cross-reactivity,consistent with the FQ-PCR kit test results.The kit is simple,portable low cost,high sensitivity and specificity,the experimental data and automated processes for large-scale field testing and monitoring.
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