摘要
目的构建用于阿尼昂尼昂热核酸检测的病毒样颗粒质控品,并对其进行评价。方法体外合成MS2噬菌体和阿尼昂尼昂病毒相关基因,通过酶切连接的方式构建表达载体,IPTG诱导表达并经Co柱纯化获得病毒样颗粒,同基质混合后制成质控品,采用荧光RT-PCR方法对质控品的均匀性、运输稳定性、保存稳定性和冻融稳定性进行系统评价。结果成功构建pET-MS2-ONNV表达载体并获得了纯化的病毒样颗粒。制备的质控品无质粒DNA污染,均匀性测试时变异系数为1.25%,往返运输后Ct值变化不明显,可在4、-20、-70℃条件下保存至少30 d,并可经受8次反复冻融。结论本研究获得了均一、稳定、耐冻融的阿尼昂尼昂热核酸检测病毒样颗粒质控品,对阿尼昂尼昂热的防控具有重要意义,也为其它类似传染病病原体的质控品构建和评价提供了一个可行的方法。
Objective To construct and evaluate O’nyong-nyong virus-like particles for quality control purpose.Methods Both MS2 phage gene and O’nyong-nyong virus gene were synthesized and ligated to construct expression vector.Virus-like particles were obtained by Co column purification after IPTG induction.Quality control products were prepared by mixing virus-like particles with normal human serum and were then evaluated using fluorescent RT-PCR focusing sample homogeneity and stability during long term shipment,storage,and repeated freeze-thaw cycles.Results Expression vector pET-MS2-ONNV and virus-like particles were obtained successfully.The quality control products prepared were free of plasmid DNA contamination with coefficient of variation of 1.25%during homogeneity evaluation.Stability evaluation indicated that these quality control products could stand long term shipment,different storage conditions and eight freezethaw cycles.Conclusion Homogeneous and stable quality control products for O’nyong-nyong fever detection was obtained and evaluated,which would play a vital role in the prevention and control of O’nyong-nyong fever.Moreover,the current study would provide a promising platform for the construction and evaluation of quality control products for other pathogens.
作者
汪海波
赵俊华
陈新彬
苏影
莫秋华
涂承宁
杨泽
WANG Hai-bo;ZHAO Jun-hua;CHEN Xin-bin;SU Ying;MO Qiu-hua;TU Cheng-ning;YANG Ze(Zhuhai International Travel Healthcare Center,Zhuhai,Guangdong 519020,China)
出处
《热带医学杂志》
CAS
2019年第9期1078-1081,共4页
Journal of Tropical Medicine
基金
国家质检总局科技计划项目（2017IK224）
海关总署科技计划项目（2018IK047）
作者简介
汪海波(1983-),男,博士,副主任技师,主要从事传染病检测与防控工作;通讯作者:杨泽,E-mail:zhithc6@163.com
