肠道集聚性大肠埃希氏菌标准物质的研制  

Development of plasmid DNA reference material of enteroaggregative Escherichia coli

作　　者：马丽侠[1] 胡雅琴[1] 蒋子敬 林华 叶德萍[1] 易艳 汤江文[1] 姜展樾 周李华[1] MA Lixia;HU Yaqin;JIANG Zijing;LIN Hua;YE Deping;YI Yan;TANG Jiangwen;JIANG Zhanyue;ZHOU Lihua(National Institute of Measurement and Testing Technology,Chengdu 610021,China;Chengdu Customs Technology Center,Chengdu 610041,China)

机构地区：[1]中国测试技术研究院,四川成都610021 [2]成都海关技术中心,四川成都610041

出　　处：《中国测试》2024年第S2期116-122,共7页China Measurement & Test

基　　金：国家重点研发计划项目(2021YFF0701905)

摘　　要：研制了3种肠道集聚性大肠埃希氏菌质粒DNA标准物质,选取肠道集聚性大肠埃希氏菌中的毒力基因ast A、agg R、pic分别构建质粒DNA标准物质,采用数字PCR技术对质粒DNA进行均匀性评估及不同温度下(-20℃,4℃,25℃,37℃)的短期(14 d)和-20℃长期(24个月)稳定性检验,联合10家实验室定值。结果显示,研制的致泻大肠埃希氏菌ast A、agg R、pic质粒DNA标准物质均匀性良好,在-20℃温度下可以储存24个月;定值结果:ast A拷贝数浓度为(2.29±0.36)×10^(7) copies/μL;agg R拷贝数浓度为(2.13±0.33)×10^(7) copies/μL;pic拷贝数浓度为(2.35±0.37)×10^(7) copies/μL。研制的标准物质适用于微生物肠道集聚性大肠埃希氏菌ast A、agg R、pic定量检测、方法评价和定性检测中的阳性对照。Three plasmid DNA reference materials of Enteroaggregative Escherichia coli were developed,the astA、aggR、pic genes were selected as targets to construct plasmid standards.They were verified by sequencing.In this study,the homogeneity and short-term(fourteen days,-20℃,4℃,25℃,37℃)and long-term(six months,-20℃)stabilities of plasmid DNA were evaluated using digital PCR.Ten laboratories were combined for value assessments of reference materials by digital PCR.The obtained experimental results show that the reference material has good homogeneity and the valid period is at least 24 months(-20℃).The calculated average of all independent measurement results(10 laboratories)was assigned as the certified value.The DNA copy number of astA was 2.29×10^(7) copies/μL with an expanded uncertainty of 0.36×10^(7) copies/μL(k=2);the DNA copy number of aggR was 2.13×10^(7) copies/μL with an expanded uncertainty of 0.33×10^(7) copies/μL(k=2);the DNA copy number of pic was 2.35×10^(7) copies/μL with an expanded uncertainty of 0.37×10^(7) copies/μL(k=2).The plasmid reference material can be used for the quantitative detection,methodology establishment and positive control for astA,aggR,pic.

关 键 词：质粒DNA 标准物质 数字PCR 大肠埃希氏菌 

分 类 号：TS201.3[轻工技术与工程—食品科学] TQ421.31[轻工技术与工程—食品科学与工程]