牛结节性皮肤病病毒（NMG 株）基因组 DNA 标准物质的研制

苏佳，白洪旭，赵炜，王德丽，刘伟洁，王芳，薛青红，陈晓春

Abstract

为提高牛结节性皮肤病病毒（LSDV）核酸检测结果准确性，本研究经病毒核酸提取、质量检定与分装，制备 LSDV NMG 株基因组 DNA 标准物质。采用荧光定量 PCR（qPCR）筛查外源病毒，评定样品纯度；运用微滴式数字 PCR（ddPCR）检测 ORF101 基因拷贝数，开展均匀性、稳定性考察，并联合 9 家实验室完成协同定值；同时借助 qPCR 开展实际应用验证。结果表明，标准物质无外源病毒污染；组内、组间检测结果无显著差异，均匀性良好。样品在 – 20 ℃可稳定保存 6 个月，4 ℃可稳定存放 7 d，25 ℃条件下稳定 24 h。该标准物质 ORF101 基因定值为 (8.6 ± 1.3) × 10³ 拷贝 /μL，适用于 qPCR 检测。综上，该标准物质定值精准、均匀性优异，可用于 LSDV 核酸检测质量管控、仪器校准与方法评价，也可为体外诊断试剂研发提供基础材料。

In order to improve the accuracy of lumpy skin disease virus (LSDV) nucleic acid detection, genomic DNA reference material (RM) of the LSDV NMG strain was prepared via nucleic acid extraction, quality verification, and aliquoting. Exogenous virus detection was performed to verify the purity of the RM. A droplet digital PCR (ddPCR) method was used to evaluate the homogeneity and stability of the RM, and collaborative quantification was completed by 9 laboratories. The applicability of the RM was validated by quantitative fluorescence PCR. No exogenous viral contamination was detected in the prepared RM. Homogeneity verification showed no significant intra-assay and inter-assay differences, indicating excellent homogeneity. Stability tests demonstrated that the RM could be stably stored at -20 ℃ for 6 months, 4 ℃ for 7 days, and 25 ℃ for 24 hours. The certified concentration of the ORF101 gene of the RM was (8.6 ± 1.3) × 10³ copies/μL, which was applicable to quantitative fluorescence PCR detection. In conclusion, the developed RM with accurate quantification and favorable homogeneity can be used for quality control, instrument calibration, and methodological evaluation of LSDV nucleic acid detection. It also provides a reliable material basis for the research and development of in vitro diagnostic reagents for LSDV.