散装食品中致病菌与卫生细菌指标检测技术研究

本研究旨在利用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）快速检测并鉴定散装即食食品中的沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌3种食源性致病菌。通过对点样方式、前处理方式、不同培养基及培养时间等各项实验条件的优化筛选确定最佳方法，并考察其检测限和稳定性，同时用所建立的方法与多重PCR法、VITEK 2等生化鉴定法进行结果比对，比较三者鉴定结果的一致性，最后进行批量食品样本检测来探寻MALDI-TOF MS鉴定法在散装即食食品中的适用性。结果表明，三种目标菌同一批次及不同传代次数的鉴定得分均大于95%，变异系数均小于2%，所建立的方法重复性和稳定性良好，且检测限为10⁵ CFU/mL，MALDI-TOF MS菌株鉴定结果与多重PCR法、生化鉴定法结果高度一致，且比二者检测用时更短，更加特异高效；在点样方式上，相较于夹心法与混合干滴法，三种覆盖法点样平均鉴定得分更高，均为99.9%，其中1 µL+1 µL覆盖法点样可获得的平均峰数目更多，特征峰更复杂且强度更高；而在前处理方式上，甲酸乙腈法与其他两种菌体处理方法相比可获得更多的出峰量和更高的峰强度，平均鉴定得分均在98%以上，且与数据库中图谱匹配度更高。综上所述，MALDI-TOF MS法在致病菌鉴定方面不仅灵敏特异、准确快速，且操作简便、结果直观，作为国标法的有力补充，为食源性致病菌的分析鉴定与分型溯源提供了新思路。

Study on detection technology of pathogenic bacteria and sanitary bacteria in bulk food

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) diagnostic tool was used to rapidly detect and identify three foodborne pathogens, Salmonella, Staphylococcus aureus and Bacillus cereus in ready-to-eat bulk foods in this study. The best method was identified by optimizing and screening various experimental conditions such as spot sampling method, pre-treatment method, different media and culture time, and its detection limit and stability were evaluated. Meanwhile, the established method was compared with multiplex PCR and the other biochemical identification methods such as VITEK 2 to contrast the consistency of the three identification results. Finally, batch food sample testing was carried out to explore the applicability of using MALDI-TOF MS to test ready-to-eat bulk foods. Results indicated that all the identification scores of the three bacteria were higher than 95% for the same batch and different passages, with a coefficient of variation of less than 2%. It demonstrated that the established method had good repeatability and stability, and the detection limit was 10⁵ CFU/mL. The results of strain identification by MALDI-TOF MS were highly consistent with those of multiplex PCR and biochemical identification methods. Moreover, the MALDI-TOF MS method was more specific and efficient. Regarding spot sampling, the three covering methods showed higher average identification scores than the sandwich and mixed methods, all of which scored 99.9%. The 1 μL+1 μL covering method spot sampling approach obtained higher average peak numbers, and more complex and stronger characteristic peaks. Regarding pre-treatment, the formic-acid-acetonitrile method obtained a higher peak volume and peak intensity than other bacterial treatment methods, with an average identification score of 98% or higher. At the same time, the matching degree with the database spectrum was higher. In summary, MALDI-TOF MS method was not only sensitive, specific, accurate and rapid in foodborn pathogen identification, but also had simple operation and intuitive in results. As a powerful supplement to the national standard method, it provided a viable alternative for analysing, identifying and typing foodborne pathogens.