非洲马瘟病毒(AV1311株)核糖核酸标准物质的研制

Development of nucleic acid reference material for African horse sickness virus(strain AV1311)

标准物质是分子生物学诊断技术的金标准。针对国内无非洲马瘟病毒标准物质的现状,以其AV1311株为原料,通过病毒培养、灭活、分装、特性检验等,成功制备非洲马瘟病毒(AV1311株)核糖核酸标准物质。同时,建立数字PCR方法,对标准物质进行均匀性评估、稳定性考察及9家实验室联合定值,并进行临床试用。结果显示,组内和组间无统计学差异,样品均匀;-20℃保存可稳定6个月,4℃保存可稳定7 d,25℃保存可稳定24 h,可冻融5次,需干冰运输;标准物质认定值为(6.9±1.1)×10^(3)copis/μL;临床试用显示,所制备的标准物质与临床样品互通性良好;最终通过全国标准物质管理委员会评审,获得国家二级标准物质证书[GBW(E)091272]。综上,本标准物质定值准确,均匀性好,量值稳定,为做好国内非洲马瘟防范工作提供了物质基础。

Reference materials (RMs) are the gold standard for molecular diagnosis technology.In view of the lack of African horse sickness virus (AHSV) RMs in China,AHSV (AV1311 strain) nucleic acid RM (NARM) was successfully prepared using AV1311 strain as raw material by virus culture,inactivation,packaging,and characteristic testing.At the same time,a digital PCR method was established to evaluate the homogeneity,stability,and quantification of the NARM in collaboration with 9 laboratories,and carried out clinical trials.The results of homogeneity evaluation showed there was no statistical differences within groups and between groups,and the samples were homogeneous.The stability investigation revealed that the NARM could be stored at-20℃for 6 months,4℃for 7 days,and at 25℃for 24hours.It could undergo up to 5 freeze-thaw cycles and requires transport on dry ice.The certified value of the NARM was(6.9±1.1)×10^(3)copies/μL.Clinical trials demonstrated that the prepared NARM had good interoperability with clinical samples.In conclusion,the prepared NARM has accurate quantification,good homogeneity and stable values,which had been successfully evaluated by National Administrative Committee[GBW(E)091272]and provides a material basis for the prevention of AHS in China.