O型口蹄疫病毒核酸检测标准样品的制备  

Preparation of O Type FMDV Reference Materical Used for Nucleic Acid Detection

作　　者：孙涛[1] 梁成珠[1] 邓明俊[1] 郑小龙[1] 王群[1] 雷质文[1] 曹丙蕾[2] 凌宗帅[2] 

机构地区：[1]山东出入境检验检疫局,山东青岛266002 [2]济南出入境检验检疫局,山东济南250014

出　　处：《动物医学进展》2014年第2期45-50,共6页Progress In Veterinary Medicine

基　　金：国家质检总局科技项目(2011IK010);国家科技支撑计划项目(2012BAK11B04)

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％）14 d,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士0.485）×108 copies,可用作O型FMDV通用核酸检测的标准质控品.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 3D gene, 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 RT- PCR 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 sta- ble at room temperature （20 C-25 C） for 2 weeks, 2 C-8 C for 3 months and --20 C for 1 year. The reference material was valued with（1. 2604-0. 485） 108 copies and can be used as positive standard FMDV sample for nucleic acid amplification testing

关 键 词：定值 O型口蹄疫病毒 实时定量RT-PCR 标准样品 

分 类 号：S852.659.6[农业科学—基础兽医学]