非洲马瘟病毒（AV1311 株）核糖核酸标准物质的研制

苏佳，张敏，白洪旭，张兵，赵炜，薛青红，杨承槐，陈晓春

Abstract

标准物质是分子生物学诊断技术的金标准。针对国内暂无非洲马瘟病毒标准物质的现状，以 AV1311 毒株为原料，经病毒培养、灭活、分装、特性检验等流程，成功研制非洲马瘟病毒（AV1311 株）核糖核酸标准物质。同时建立数字 PCR 检测方法，开展均匀性评估、稳定性考察、9 家实验室联合定值及临床试用。结果显示，组内、组间检测结果无统计学差异，样品均匀性良好；-20 ℃条件下可稳定保存6 个月，4 ℃可稳定保存7 d，25 ℃可稳定存放24 h，耐受5 次冻融，样品需干冰运输；标准物质认定值为 (6.9 ± 1.1) × 10³ copies/μL；临床试用证实该标准物质与临床样品互通性优异。该物质已通过全国标准物质管理委员会评审，获批国家二级标准物质证书 [GBW (E) 091272]。综上，本标准物质定值精准、均匀性与稳定性俱佳，为国内非洲马瘟防控筑牢物质基础。

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 clinical trials were conducted. The results of homogeneity evaluation showed there were 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 25 ℃ for 24 hours. 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) × 10³ 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 has been successfully approved by National Administrative Committee [GBW (E) 091272] and provides a material basis for the prevention of AHS in China.