病毒性神经坏死病病毒核酸检测用标准物质的制备

Preparation of Reference Materials Used for Detecting Nucleic Acids of Nervous Necrosis Virus

利用基因克隆和体外转录技术,制备病毒性神经坏死病病毒(Nervous necrosis virus,NNV)核酸检测标准物质。设计NNV RNA2基因的特异性引物,通过RT-PCR获得目的片段并连接至p GEM-T载体;采用体外转录方法,获得大量纯品RNA。初步定量稀释至约10~8 copies/μL,均匀性检验结果显示样品间差异<5%;稳定性检验表明,室温(20~25℃)保存7 d、冷藏(2~8℃)保存1个月和冷冻(-20℃)保存6个月的含量均无明显变化。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,并根据委托单位的定值结果进行不确定度估算。核酸标准物质定值为(8.452±0.068)×10~8 copies/μL,可用作NNV核酸检测的标准质控品。

In this article,the reference materials for detecting nucleic acids of Nervous Necrosis Virus（NNV）were prepared by technologies of gene cloning and transcription in vitro. Firstly,the specific primers were designed based on the sequence of RNA2 gene,the target fragments were obtained by RT-PCR and cloned into pGME-T vector. Then large amounts of pure RNA were prepared by transcription in vitro. After quantitative dilution,the concentration of RNA materials was about 108 copies/μL. Results of homogeneity test showed the difference among the RNA materials was less than 5%. For the stability test,results showed there was no obvious change when the RNA materials were under the conditions below：room temperature（20-25 ℃）,preserved for 7 days;cold storage（2-8 ℃）,preserved for 1 month;refrigeration（–20 ℃）,preserved for 6 months. Next,the RNA materials were valued by concentration determination of nucleic acids and conversion of nucleic acid copies,and estimation of uncertainty was carried out. As a result, the RNA materials could be used as the standard control materials and the determined value was （8.452±0.068）×10 8 copies/μL.