肠道出血性大肠埃希菌菌株及其基因组DNA标准物质的研制及评价

Preparation and evaluation of reference of enterohemorrhagic Escherichia coli strains and genomic DNA

目的 筛选肠道致病性大肠埃希氏菌(enterohemorrhagic Escherichia coli,EHEC)菌株,并将其申报为中国医学细菌保藏管理中心(China Medical Bacterial Species Conservation and Management Center,CMCC)标准菌株;以此标准菌株研制我国自主知识产权的EHEC菌株及其基因组DNA标准物质,并将其申报为国家药品标准物质。方法 依据GB4789.6-2016《食品安全国家标准食品微生物学检验致泻大肠埃希氏菌检验》,从160株来源于腹泻患者的大肠埃希菌中筛选EHEC菌株,按照CMCC菌株管理规定整理材料将其申报为标准菌株。用其制备EHEC菌液及其基因组DNA溶液,采用冷冻干燥技术分别制备菌株类(103CFU/样品)和基因组DNA类(20 ng/样品)样品各600个。将这两种样品各随机抽取20个进行均匀性检验;并分别于25、37℃条件下存放1、3、5、7 d取样检测运输稳定性,于4℃条件下存放7、14、28 d取样检测短期保存稳定性,于-20℃条件下存放14、28、60 d取样检测长期保存稳定性。组织3家实验室对两种样品进行协同标定。筛选20种食品基质对菌株样品进行使用效果评价。结果 从160株来源于患者的大肠埃希菌中筛选出1株EHEC菌株,最终申报为CMCC(B)43207标准菌株。均匀性检验中,F_(菌株样品)=0.662 <F_(INV)(0.05,19,20),P> 0.05;20个基因组样品的eae、stx1、stx2基因均为阳性。于25和37℃保存7 d、-20℃保存60 d、4℃保存28 d后,菌株样品活菌含量均为10^(3)CFU/样品,基因组样品的eae、stx1、stx2基因均为阳性。随机抽取的EHEC菌株和基因组DNA样品经3家实验室鉴定均为EHEC,活菌含量均在10^(3)CFU/样品的水平,且eae、stx1、stx2基因均为阳性。在20种食品基质中加入样品后均可检出,本底对照均未检出。EHEC菌株及其基因组DNA样品均纳入我国药品标准物质,编号分别为80024和80056。结论 研制的具有自主知识产权的EHEC菌株及其基因组DNA标准物质可提高EHEC检验的时效性,弥补了我国此方面空白。

Objective To screen enterohemorrhagic Escherichia coli(EHEC) strain and declare it as a standard strain of China Medical Bacterial Species Conservation and Management Center(CMCC).On the base,to prepare strain reference and genomic DNA reference of EHECand declare them as national drug reference with independent intellectual property rights in our country.Methods According to GB4789.6-2016 National Food Safety Standards-Food Microbiology TestDiarrheagenic Escherichia coil Test,the EHEC strain was screened from 160 Escherichia coli strains from patients with diarrhea and declared as a standard strain of CMCC according to management regulations.EHEC bacterial solution and genomic DNA solution were prepared,and freeze-drying technology was used to prepare 600 strain(10~3 CFU/sample) and genomic DNA(20 ng/sample) samples respectively.20 strain and 20 genomic DNA samples were randomly selected for uniformity test.Samples storing at 25 ℃ and 37 ℃ for 1,3,5 and 7 d were taken respectively to test the transportation stability.Then the samples were tested for the short-term storage stability by storing at 4 ℃ for 7,14 and 28 d,and for the long-term storage stability by storing at 20 ℃ for 14,28 and 60 d.Three laboratories were organized for collaborative calibration.20 food products were chosen as the substrate to evaluate the application effect of strain samples.Results The one EHEC strain selected from 160 Escherichia coli strains from patients was finally declared as the CMCC(B) 43207standard strain.In the uniformity test,F_(strainsample)=0.662 F_(INV)(0.05,19 and 20),P 0.05,and eae,stxl and stx2 of 20genomic samples were all positive.After storage at 25 and 37 ℃ for 7 d,-20 ℃ for 60 d and 4 ℃ for 28 d,the viable bacteria content of the strain samples was still 103 CFU/sample,and eae,stxl and stx2 of the genomic samples were positive.EHEC strains and genomic DNA samples selected randomly were identified as EHEC by three laboratories,the viable bacteria content was 10~3 CFU/sample,and eae,stxl and stx2 were all positive.It was detected in 20 kinds of food substrates after adding samples,but not in the background control.EHEC strain and the genomic DNA sample were included in drug standard materials in our country,numbered 80024 and 80056,respectively.Conclusion The prepared EHEC strain and genomic DNA standard materials with independent intellectual property rights can improve the timeliness of EHEC testing and make up for the gap in our country.