创伤弧菌核酸检测试剂国家参考品的建立
Establishment of a national reference panel for Vibrio vulnificus nucleic acid detection kits
赵兰青 1刘红 2邓明镜 3麻婷婷 1许四宏1
作者信息
- 1. 中国食品药品检定研究院传染病诊断二室,北京 100050;国家药品监督管理局体外诊断试剂质量研究与评价重点实验室,北京 100050
- 2. 中国人民解放军联勤保障部队第九八三医院采购管理科,天津 300142
- 3. 北京大学生命科学学院,北京 100091
- 折叠
摘要
目的 建立创伤弧菌核酸检测试剂国家参考品并制定质量标准,用于该类试剂的质量评价.方法 收集培养多种创伤弧菌及其他弧菌病原体,经菌落鉴定、16s rRNA测序分析及实时荧光定量PCR试剂检测后筛选出18份样本,稀释、分装后组成创伤弧菌核酸检测试剂国家参考品.邀请不同的实验室对参考品进行协作标定,并对参考品进行均匀性和稳定性考察.结果 建立的国家参考品,包括8份阳性参考品P1～P8、10份阴性参考品N1～N10、1份重复性参考品R和1份检测限参考品L;L使用菌落计数法测定浓度为1×108 CFU/mL.4家实验室参与了国家参考品的协作标定,并根据结果制定质量标准为阳性符合率8/8,阴性符合率10/10,检出限要求至少为1×103 CFU/mL及以上阳性,重复性要求检测10次均为阳性,且Ct值或定量值变异系数不大于5.0%.国家参考品均匀性检测的Ct值变异系数均不高于5%,符合要求;在2～8℃冷藏条件、室温放置7天,均未影响参考品稳定性.结论 成功建立了创伤弧菌核酸检测试剂国家参考品,可用于企业试剂研发的质量控制及评价.
Abstract
Objective To establish a national reference panel for Vibrio vulnificus nucleic acid de-tection reagents and set standards,which aimed to evaluate the quality of related kits.Methods A variety of Vibrio vulnificus and other Vibrio pathogens were collected and cultured.After colony identification,16s rRNA sequencing analysis and detection of a real-time fluorescence quantitative PCR reagent,18 samples were selected,diluted,and packaged to form a national reference panel for Vibrio vulnificus nucleic acid detection kits.Different laboratories were invited to coordinate calibration of the panel,and the uniformity and stability were further investigated.Results The established national reference panel included 8 positive references P1～P8,10 negative references N1～N10,1 repetitive reference R and 1 limited detection reference L;Refer-ence L was determined the concentration of 1×108 CFU/mL by colony-counting methods.Four laboratories par-ticipated in the collaborative calibration of national reference materials and developed quality standards based on the results:positive coincidence rate of 8/8,negative coincidence rate of 10/10;the detection limit is at least to be 1×103 CFU/mL,the repeatability is required to be positive for 10 times,and the variation coefficient of Ct value or quantitative value is not more than 5.0%.The coefficient of variation of Ct value in uniformity de-tection of a national reference was less than 5%,which met the requirements.The stability of the reference prod-uct was not affected when it was refrigerated at 2～8℃and room temperature for 7 days.Conclusion The nation-al reference panel for vibrio vulnificus nucleic acid detection kits was successfully established,which can be used for quality control and evaluation for enterprises which planned to develop and research related kits.
关键词
创伤弧菌/国家参考品/核酸检测/质量评价Key words
Vibrio vulnificus/National references/Nucleic acid detection/Quality evaluation引用本文复制引用
基金项目
&&(2018ZX10102001-002-002)
出版年
2024
NETL
NSTL
维普
万方数据