摘要
为提高牛结节性皮肤病病毒(LSDV)核酸检测结果的准确性,本研究通过病毒核酸提取及质量检测、分装,制备LSDV NMG株基因组DNA标准物质。通过荧光定量PCR(qPCR)检测外源病毒评价其纯净性;采用液滴式数字PCR(ddPCR)方法检测LSDV ORF101基因的拷贝数以评估该标准物质的均匀性、稳定性并与9家实验室协作标定;进一步采用qPCR方法对该标准物质试用。结果显示,本研究制备的标准物质不存在外源病毒污染;组内和组间无显著性差异,均匀性好;标准物质于-20℃稳定保存6个月,4℃稳定保存7d,25℃稳定保存24 h;标准物质ORF101基因最终定值为(8.6±1.3)×10~3拷贝/μL;可以用于qPCR检测。综上,该标准物质定值准确,均匀性好,不仅可以用于LSDV核酸检测相关实验的质量控制、仪器校准、检测方法评价,还可为研制体外诊断试剂提供物质基础。
In order to improve the accuracy for the nucleic acid detection of lumpy skin disease virus(LSDV),we prepared a genomic DNA reference material(RM)for LSDV(NMG strain)through steps including virus nucleic acid extraction,quality detection and aliquoting.The purity of the RM was evaluated through exogenous virus detection.The homogeneity,stability,and quantification of the RM were assessed using droplet digital PCR(dd PCR)assay in 9 collaborating laboratories.The RM was applied on a fluorescence quantitative PCR detection.The results of exogenous virus detection indicated no contamination available in the RM.The results of homogeneity assessment showed no statistically significant differences for both the intra-assay and inter-assay,demonstrating a good homogeneity.The stability investigation revealed that the RM can be stored at-20℃for 6months,4℃for 7 days,and 25℃for 24 hours.The certified value for the ORF101 gene of the RM was determined as(8.6±1.3)×10~3 copies/μL.The RM can be applied for the testing with the fluorescence quantitative PCR.In conclusion,this RM has accurate certified value,good homogeneity,making it applicable for the quality control in laboratories conducting LSDV nucleic acid testing,instrument calibration,and detection methods evaluation.Furthermore,it provides a foundation for the efficient development of in vitro diagnostic reagents.
作者
苏佳
白洪旭
赵炜
王德丽
刘伟洁
王芳
薛青红
陈晓春
SU Jia;BAI Hong-xu;ZHAO Wei;WANG De-li;LIU Wei-jie;WANG Fang;XUE Qing-hong;CHEN Xiao-chun(China Institute of Veterinary Drug Control,Beijing 100081,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第3期320-325,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划项目(2021YFD1800500)。
