弯曲杆菌16S rRNA基因质粒DNA标准物质的研制  

Development of Plasmid DNA Reference Material of Campylobacter 16S rRNA

作　　者：张喜悦[1] 张铭洋 赵格[1] 曲志娜[1] 刘俊辉[1] 王娟[1] 赵建梅[1] 高玉斌 黄秀梅[1] 刘娜[1] 程慧敏 徐佳微 邹明 王君玮[1] Zhang Xiyue;Zhang Mingyang;Zhao Ge;Qu Zhina;Liu Junhui;Wang Juan;Zhao Jianmei;Gao Yubin;Huang Xiumei;Liu Na;Cheng Huimin;Xu Jiawei;Zou Ming;Wang Junwei(China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China;College of Animal Medicine,Qingdao Agricultural University,Qingdao,Shandong 266000,China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]青岛农业大学动物医学院,山东青岛266000

出　　处：《中国动物检疫》2023年第7期103-109,共7页China Animal Health Inspection

基　　金：国家重点研发计划项目(2022YFD1301003,2022YFC2303900);山东省重点研发计划项目(2022CXGC010606-01-05);中国动物卫生与流行病学中心创新基金项目(DW2021001-13)。

摘　　要：弯曲杆菌引起的食源性胃肠道疾病是21世纪人类面临的一项严重经济和公共卫生负担。当前国内外进行的弯曲杆菌属分子生物学检测方法研究的靶基因均为16S rRNA基因。为满足检测试剂标准化需求,制备了候选弯曲杆菌16S rRNA基因质粒DNA标准物质,经均匀性和稳定性检验,证实其于-20℃保存12个月,4℃短期保存8 d,反复冻融30次后依然稳定。对候选标准物质采用微滴式数字PCR(ddPCR)方法,联合8家实验室进行联合定值,对不确定度进行量化、合成,确定了标准物质的扩展不确定度。最终确定弯曲杆菌16S rRNA质粒DNA标准物质标准值为4.95×10^(3)copies/μL,扩展不确定度(k=2)为0.47×10^(3)copies/μL。临床试用结果表明,该产品具有良好的均匀性和稳定性。该标准物质的研制,为我国弯曲杆菌分子生物学检测试剂的标准化奠定了基础。Food-borne gastrointestinal diseases caused by Campylobacter have become a serious economic and public health burden for human in the 21st century.16S rRNA gene has been currently researched as the target gene for Campylobacter molecular biological detection.In order to standardize detection reagents,candidate plasmid DNA reference material of Campylobacter 16S rRNA was prepared and confirmed to be stable through homogeneity and stability tests when it was stored for 12 months at-20℃,or 8 days at 4℃,after 30 repeated freeze-thaw cycles.The candidate reference material was valued together by 8 laboratories by droplet digital PCR assay(ddPCR),and its uncertainty was quantified and synthesized to determine its extended uncertainty.The standard value of the reference material was 4.95×10^(3)copies/μL,and the expanded uncertainty(k=2)was 0.47×10^(3)copies/μL.Clinical trial results indicated that the reference material had good homogeneity and stability.The development of the reference material laid a foundation for the standardization of Campylobacter molecular biological detection reagents.

关 键 词：弯曲杆菌 16S rRNA基因 标准物质 均匀性 稳定性 可重复性 

分 类 号：Q93-331[生物学—微生物学]