中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > O型口蹄疫病毒核酸检测标准样品的制备 动物医学进展 2014,35 (2) 45-50
Position: Home > Articles > Preparation of O Type FMDV Reference Materical Used for Nucleic Acid Detection Progress in Veterinary Medicine 2014,35 (2) 45-50

O型口蹄疫病毒核酸检测标准样品的制备

作  者：
孙涛;梁成珠;邓明俊;郑小龙;王群;雷质文;曹丙蕾;凌宗帅
单  位：
济南出入境检验检疫局;山东出入境检验检疫局
关键词：
定值;O型口蹄疫病毒;实时定量RT-PCR;标准样品
摘  要：
利用基因克隆和体外转录技术制备O型口蹄疫病毒(FMDV)核酸检测标准样品。设计O型FMDV 3D基因的全长开放阅读框的克隆引物,RT-PCR获得相应片段,连接至PGEM-T载体,测序后采用体外转录方法制备RNA纯品,初步定量稀释后,混合分装作为核酸标准样品候选物。采用实时荧光定量RT-PCR方法进行均匀性和稳定性检验,并委托外部实验室采用外标实时定量RT-PCR方法对转录的RNA片段进行定值。通过绘制荧光定量标准扩增曲线计算标准物质含量(拷贝数),并根据委托单位的定值结果进行不确定度的估算。均一性结果显示瓶间差异小于5%,稳定试验表明室温20℃~25℃(相对湿度20%~50%)14d,2℃~8℃3个月及-20℃保存1年的含量均无明显变化。核酸标准物质定值为(1.260±0.485)×108 copies,可用作O型FMDV通用核酸检测的标准质控品。
译  名：
Preparation of O Type FMDV Reference Materical Used for Nucleic Acid Detection
作  者：
SUN Tao;LIANG Cheng-zhu;DENG Ming-jun;ZHENG Xiao-long;WANG Qun;LEI Zhi-wen;CAO Bing-lei;LING Zong-shuai;Shandong Entry-exit Inspection and Quarantine Bureau;Jinan Entry-exit Inspection and Quarantine Bureau;
关键词：
metrology;;Food-and-mouth disease virus type O;;real-time RT-PCR;;reference material
摘  要：
Using gene cloning and in vitro transcription technology,food-and-mouth disease virus type O positive reference material was preparaed for nucleic acid amplification testing.The primers were designed to amplify the complete ORFs of 3Dgene,and obtain the corresponding fragment by RT-PCR,and then cloned into pGME-T vector and sequenced.The pure RNA were prepared by transcription in vitro and quantitatively diluted as nucleic acid candidate.After aliquot,the homogenity and stability testing were conducted using real-time RT-PCR.With the standard curves were constructed,the gene copies of F genes were determined and uncertainty estimates were made by commissioned laboratories using real-time RTPCR by adding external standard substance.The results showed that difference between groups were less than 5% by homogeneity testing.The stability test indicated that the prepared reference material was stable at room temperature(20℃-25℃)for 2weeks,2 ℃-8 ℃for 3months and-20 ℃for 1year.The reference material was valued with(1.260±0.485)×108 copies and can be used as positive standard FMDV sample for nucleic acid amplification testing

相似文章

导出引用
计量
文章访问数: 10
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46