病毒性出血性败血症病毒RNA标准物质的研制  

Preparation of VHS Reference Materical Used for Nucleic Acid Detection

作　　者：刘志玲[1] 陈茹[1] 朱道中[1] 吴晓薇[1] 林志雄[1] 田纯见[1] 罗琼[1] 段燕喻[1] LIU Zhi-ling;CHEN Ru;ZHU Dao-zhong;WU Xiao-wei;LIN Zhi-xiong;TIAN Chun-jian;LUO Qiong;DUAN Yan-yu(Inspection and Quarantine Technology Center, Guangdong Entry-Exit Inspection and Quarantine Bureau/Guangdong Key Laboratory of Import and Export Technical Measures of Animal, Plant and Food, Guangzhou 510623, China)

机构地区：[1]广东出入境检验检疫局检验检疫技术中心广东省动植物与食品进出口技术措施研究重点实验室,广东广州510623

出　　处：《中国兽医杂志》2018年第3期15-19,共5页Chinese Journal of Veterinary Medicine

基　　金：国家质检总局科技项目(2016IK047)

摘　　要：利用基因克隆和体外转录技术制备病毒性出血性败血症病毒(VHSV)核酸检测标准物质。设计VHSV N基因的克隆引物,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物质候选物。采用核酸浓度测定与拷贝数换算的方法对转录的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%,稳定性试验表明室温20℃~25℃(相对湿度20%~25%)7 d,2℃~8℃保存1个月及-20℃保存6个月的含量均无明显变化。核酸标准物质定值为(6.625±0.149)×10~8copies/μL,可用作VHSV核酸检测的标准质控品。Positive reference material of VHSV was established for nucleic acid amplification testing. The primers were designed to amplify the N gene,the corresponding fragment was obtaind by RT-PCR,and then cloned into p GME-T vector and sequenced. The pure RNAs were prepared by transcriptionin vitro and quantitatively diluted as nucleic candidate. After aliquot,the homogeneity and stability testing were conducted using real-time RT-PCR. With the standard curves were constructed,the gene copies of N genes were determined and uncertainty estimates were made by commissioned laboratories. The results showed that difference between groups was less than 5% by homogeneity testing. The stability test indicated that the prepared reference material was stable at room temperature（ 20 ℃ ~ 25 ℃） for 1 week,2 ℃ ~ 8 ℃ for 1 months and-20 ℃ for 6 months. The reference material was valued with（ 6. 625 ± 0. 149） × 10~8 copies/μL and can be used as positive standard FMDV sample for nucleic and amplification testing.

关 键 词：病毒性出血性败血症 实时定量RT-PCR 标准物质 

分 类 号：S855.3[农业科学—临床兽医学]