单核细胞增生李斯特氏菌能力验证样品的制备与验证

Preparation and verification of Listeria monocytogenes samples for proficiency testing

目的研究制备适用于验证、评价实验室或检测机构检测能力的单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)的能力验证冷冻干燥样品。方法对目标菌与干扰菌进行10倍梯度稀释,对不同稀释度的菌液进行菌落计数得到菌含量,确定单核细胞增生李斯特氏菌与干扰菌的添加量;对保护剂和预冻条件进行筛选优化,制备单核细胞增生李斯特氏菌能力验证样品。冻干样品采用西林瓶真空包装,4℃条件下冷藏保存,随机取样检测评估样品均匀性,并在210 d期间内定期抽样检测不同贮存温度下的冻干样品,检测评估样品的稳定性。结果每个冷冻干燥阳性样品最终添加目标菌单增李斯特菌和干扰菌分别为10~2 CFU/m L和10~4 CFU/m L;每个阴性样品添加干扰菌10~4 CFU/m L;最佳冻干保护剂的组合为海藻糖3%,脱脂奶粉8%,谷氨酸钠1.5%。检测结果显示PT冻干样品具有较好的均匀性和稳定性。结论建立的单核细胞增生李斯特氏菌能力验证样品制备方法与评估程序均为有效,适用于验证与评价实验室或检查机构检测能力,满足能力验证活动的要求。

Objective To prepare proficiency testing（PT） lyophilization samples of Listeria monocytogenes（LM） which were used to verify and evaluate the testing ability of participating laboratories or inspection institutions. Methods A suitable proportion and concentration of Listeria monocytogenes and interference bacteria were selected through 10 times dilution and colony counts. After the lyoprotectant and pre-freezing conditions were optimized, Listeria monocytogenes PT samples were prepared, vacuum packaged by Schering bottles and stored at 4 ℃ cold storage. The uniformity of PT samples was evaluated through random sampling. Freeze-dried samples at different storage temperatures were periodic detected during the period of 210 d so as to evaluate the stability of PT samples. Results Totally 10~2 CFU/m L Listeria monocytogenes and 10~4 CFU/m L interference bacteria were added to every positive PT sample respectively, and 10~4 CFU/m L interference bacteria were added to every negative PT sample. The optimum combination of lyoprotectant consisted of 3% rehalose, 8% skim milk powder, and 1.5% sodium glutamate. The testing results showed that the PT freeze-dried samples had good uniformity and stability. Conclusion The preparation methods and evaluation procedures of Listeria monocytogenes PT lyophilization samples are effective, which can evaluate the testing ability of laboratories and inspection institutions, and meet the requirements of proficiency testing.