6种克罗诺杆菌精准鉴定及标准物质的制备研究

Research on accurate identification of 6 kinds of Cronobacter and reference material preparation

目的制备具有我国食源性特点的6种克罗诺杆菌检验用标准物质。方法通过生化、基质辅助激光解吸电离飞行时间质谱法(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)及二代高通量全基因组测序技术对研究用菌株进行菌种鉴定确认后,制备10^(4)CFU/样品浓度的标准物质。参照CNAS—GL003:2008《能力验证样品均匀性和稳定性评价指南》等标准要求对样品进行均匀性检验后,采用单因素方差分析对结果进行评价。将样品放于-20、4、25和37℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据GB 4789.15—2016《食品安全国家标准食品微生物学检验霉菌和酵母计数》,将研制的6种标准物质分别加入20件乳粉样品中进行检验,检测真实食品样品中适用性。结果6株菌株生化鉴定结果均为阪崎克罗诺杆菌属(Cronobacter准确度为92%~99%;MALDI-TOF MS鉴定结果均为阪崎克罗诺杆菌(Cronobacter sakazakii),分数为1.914~2.244;全基因组测序鉴定结果与菌株已知种或亚种信息一致。6种标准物质F值分别为0.422、0.922、0.980、0.409、0.721及0.631,F<F_(0.05),符合均匀性要求。25及37℃放置7 d,6种标准物质中菌含量仍保持在10^(4)CFU/样品。6种标准物质放置于4℃储存28 d及放置于-20℃储存90 d,菌含量均为10^(4)CFU/样品,且存活率均在80%以上。20件真实乳粉样品中分别加入6种克罗诺杆菌标准物质进行检验,均可以检出;经3家实验室协作标定,6种克罗诺杆菌标准物质浓度均为10^(4)CFU/样品。结论本研究制备的6种克罗诺杆菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于婴幼儿乳粉中克罗诺杆菌的检验。

Objective To prepare 6 different kinds of reference materials for the detection of Cronobacter with the characteristics of foodborne in China.Methods The strains were identified by biochemistry,matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS)and the next-generation sequencing and then 10^(4)CFU/sample concentration reference materials were prepared.According to the requirements including CNAS—GL003:2008 Guide for evaluation of homogeneity and stability of proficiency testing samples,the homogeneity of samples was tested,and the results were evaluated by one-way analysis of variance(ANOVA).The samples were placed at-20,4,25 and 37℃for transport stability test and storage stability test respectively.According to GB 4789.15—2016 National food safety standard-Microbiological examination-Molds and yeasts count,the developed 6 kinds of reference materials were added into 20 milk powder samples for testing to detect the applicability.Results All 6 strains were Cronobacter sakazakii group using biochemical identification method,with the accuracy of 92%-99%;The results of MALDI-TOF MS were all Cronobacter sakazakii with a score of 1.914-2.244;The whole genome sequencing results were consistent with the known species or subspecies information.The F values of the 6 kinds of reference materials were 0.422,0.922,0.980,0.409,0.721 and 0.631 respectively,F<F_(0.05),which met the uniformity requirements.After 7 d at 25 and 37℃,the bacterial content of the 6 kinds of reference materials remained at 10^(4)CFU/sample.When the 6 kinds of reference materials were stored at 4℃for 28 d and-20℃for 90 d,the bacterial content was 10^(4)CFU/sample,and the survival rates were more than 80%.Six kinds of Cronobacter reference materials were added to 20 real milk powder samples,and all of them could be detected.After the collaborative calibration by 3 laboratories,the concentration of 6 kinds of Cronobacter reference materials was 10^(4)CFU/sample.Conclusion The homogeneity,stability,application verification and collaborative calibration results of 6 kinds of Cronobacter reference materials prepared in this study meet the requirements,and can be applied to the detection of Cronobacter in infant milk powder.