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

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.