肠道集聚性大肠埃希氏菌菌体和gDNA标准物质的研制

Preparation of Reference Materials for Enteroaggregative Escherichia coli Cells and Genomic DNA

目的:为满足肠道集聚性大肠埃希氏菌准确检测的实验室质量控制和能力验证需求,研制具有我国自主知识产权且具有全基因组测序信息的均匀稳定的肠道集聚性大肠埃希氏菌菌体及gDNA标准物质。方法:利用二代高通量测序技术对肠道集聚性大肠埃希氏菌(CMCC 44841)进行全基因组测序,明确CMCC 44841的种属、血清分型、多位点序列分型和毒力基因。对CMCC 44841进行astA、aggR、pic特征性基因聚合酶链式反应(polymerase chain reaction,PCR)确认。采用冷冻干燥技术制备含量为103 CFU/样品的菌球和含量为20 ng/样品的gDNA小球。参照CNAS-GL017对20个菌球样品进行均匀性检验,采用单因素方差分析对结果进行统计分析。将样品分别于-20、4、25℃和37℃条件下保藏,对其贮藏稳定性和运输稳定性进行评价。组织3家实验室进行协同标定和验证。使用5种不同基质的即食食品样本,按照国标法检验标准物质的适用性。结果:利用生物信息学分析,CMCC44841基因组大小为5.057285 Mb,GC含量为50.6%,编码区基因5173个。种属鉴定结果为大肠埃希氏菌(Escherichia coli),血清预测结果为O127:H21,MLST为ST40型,携带除aggR、astA、pic之外其他相关的毒力基因。PCR扩增出astA、aggR、pic片段大小分别为102、400 bp和1111 bp。菌体标准物质均匀性检验结果F=1.59,符合标准物质的要求。菌体和gDNA标准物质样品于-20℃和4℃保藏60 d,25℃保藏7 d,37℃保藏5 d后仍然稳定。经3家实验室协同标定,菌体标准物质样品含量均为103 CFU/样品。20件不同基质的食品样品加入肠道集聚性大肠埃希氏菌标准物质进行检验,均可以检出。结论:本研究所制备的肠道集聚性大肠埃希氏菌菌体及gDNA标准物质所用菌株来源于我国国内分离菌株,且具有明确的全基因组序列信息,均匀性和稳定性均符合要求,适用性良好,能够满足食品检测实验室的质量控制和能力验证的需求。

Objective:To meet the requirements of laboratory quality control and proficiency testing in enteroaggregative Escherichia coli(EAEC)detection,homogenous and stable reference materials for enteroaggregative E.coli cells and genomic DNA(gDNA),with independent intellectual property rights,were developed.Methods:Next-generation sequencing(NGS)was used for whole genome sequencing of enteroaggregative E.coli(CMCC 44841).Bioinformatics analysis was performed on the sequencing results for species and virulence genes identification,serotyping,and multi-locus sequence typing(MLST).The characteristic genes of astA,aggR and pic were confirmed by PCR.Freeze-dried reference materials containing 103 CFU of cells and 20 ng of gDNA per sample were prepared.According to CNAS-GL017,homogeneity testing was performed on the 20 EAEC samples,and the results were statistically analyzed by one-way analysis of variance(ANOVA).The samples were stored at−20,4,25 or 37℃to evaluate their stability during storage and transportation.Then,three laboratories were organized for collaborative calibration and verification,and the applicability of the reference materials was tested on five different ready-to-eat food matrices according to the national standard method.Results:The genome size of CMCC44841 was 5.057285 Mb,the GC content was 50.6%,and the genome contained 5173 coding genes.The strain was identified as E.coli,serotyped as O127:H21,and typed as ST40 by MLST,carrying other virulence genes apart from aggR,astA and pic.The PCR fragments of astA,aggR and pic were 102,400 and 1111 bp in size,respectively.The result of homogeneity testing was F=1.59,which meets the requirements for standard materials.The EAEC cell and gDNA samples were stable at−20 and 4℃for 60 days,at 25℃for 7 days,and at 37℃for 5 days.The collaborative interlaboratory calibration confirmed the EAEC cell reference material to contain 103 CFU of cells.The reference materials could be detected when they were added to 20 sample of different food matrices.Conclusion:The EAEC cell and gDNA reference materials,prepared from isolates obtained in the country,contain clear whole genome sequence information.They have good homogeneity,stability and applicability and can meet the requirements of quality control and proficiency testing in food testing laboratories.