猪繁殖与呼吸综合征病毒MS2装甲病毒样颗粒质控品的制备及应用  

Preparation and application of PRRSV MS2 armored virus-like particles qualityas control products

作　　者：何嘉敏 庞旋飞 罗律 杨佳臻 张宝真 伍建敏 刘文娜 李中圣 白挨泉[1] HE Jiamin;PANG Xuanfei;LUO Lyu;YANG Jiazhen;ZHANG Baozhen;WU Jianmin;LIU Wenna;LI Zhongsheng;BAI Yiquan(School of Science and Technology,Foshan University,Guangdong 528231,China;Guangdong Haida Animal Husbandry&Veterinary Institute Co.,Ltd.,Guangzhou 511400,China)

机构地区：[1]佛山科学技术学院生命工程与工程学院,广东佛山528231 [2]广东海大畜牧兽医研究院有限公司,广东广州511400

出　　处：《中国兽医学报》2024年第11期2316-2323,共8页Chinese Journal of Veterinary Science

基　　金：广东省科技计划资助项目(2020B1212070023);以农产品为单元的广东省现代农业产业技术体系创新团队建设资助项目(生猪)(090/BKS209152)

摘　　要：为研制一种可以应用猪繁殖与呼吸综合征病毒(PRRSV)实时荧光定量PCR(RT-qPCR)核酸检测的阳性质控品,本研究使用MS2噬菌体装甲RNA(Armored RNA)技术制备PRRSV-1和PRRSV-2 M基因的阳性质控品。将扩增的PRRSV-1和PRRSV-2 M基因进行纯化回收,连接到内含MS2成熟酶蛋白基因与衣壳蛋白的pET28b载体,通过转化至BL21(DE3)后,经IPTG诱导表达,利用PEG6000沉淀法纯化,制备出内含PRRSV M基因装甲RNA病毒样颗粒(AR-PRRSV)。后续进行性能评估,作为PRRSV-1和PRRSV-2 M基因的阳性质控品,AR-PRRSV1M和AR-PRRSV2M使用YY/T 1652-2019标准进行计算,数据表明均匀性良好,-20、4、25℃能稳定保存60 d,37℃能稳定保存30 d。使用RT-qPCR进行初步定量后,利用数字定量PCR(ddPCR)对制备的装甲病毒样颗粒AR-PRRSV1M和AR-PRRSV2M进行定值,结果10^(4)拷贝/μL的AR-PRRSV1M和AR-PRRSV2M ddPCR定值均为(1.33±0.50)×10^(4)拷贝/μL。本研究结果表明,应用定值后的AR-PRRSVM,能够实现对检测全过程(核酸提取、反转录和RT-qPCR)的质量控制。In order to develop a positive quality control products for the detection of porcine reproductive and respiratory syndrome virus(PRRSV)nucleic acid by real-time fluorescent quantitative PCR(RT-qPCR),the positive quality control products of PRRSV-1 and PRRSV-2 M genes were prepared using armored RNA technology of MS2 phage.PRRSV-1 and PRRSV-2 M genes were amplified,purified and recovere1,and ligated into pET28b vector containing MS2 mature enzyme protein gene and capsid protein.After transformed into BL21(DE3),the gene products were induced by IPTG and purified by PEG6000 precipitation method to prepare the armored RNA viruslike particles(AR-PRRSV)containing PRRSV M gene.Following the performance evaluation,as the positive quality control products of PRRSV-1 and PRRSV-2 M genes,AR-PRRSV1M and ARPRRSV2M were calculaled using YY/T 1652-2019 standard.Results showed that it had a good uniformily,stable storage for the armored virus-like particles at-20,4,25℃for 60 d,and 37℃for 30 d.The prepared armored virus-like particles AR-PRRSV1M and AR-PRRSV2M were determined by digital quantitative PCR(ddPCR)after preliminary quantification by RT-qPCR.The10^(4) copies/μL of AR-PRRSV1M and AR-PRRSV2M ddPCR fixation was(1.33±0.50)×10^(4) copies/μL.The above results indicates that the AR-PRRSVM can be used as the quality control of the whole detection process(nucleic acid extraction,reverse transcription and RT-qPCR).

关 键 词：猪繁殖与呼吸综合征病毒 装甲RNA 核酸检测 质控品 

分 类 号：S852.65[农业科学—基础兽医学]