1株李斯特菌噬菌体的分离、保存及生物学特性研究  

Isolation,preservation,and biological characteristics analysis of a Listeria bacteriophage

作　　者：刘凌云 毛盼 陈晋妮 李玲玲 王艳[2] 宋敬东[3] 陈峥宏 叶长芸[2] LIU Ling-yun;MAO Pan;CHEN Jin-ni;LI Ling-ling;WANG Yan;SONG Jing-dong;CHEN Zheng-hong;YE Chang-yun(Department of Microbiology,School of Basic Medical Science,Guizhou Medical University,Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou Province,Guiyang 550025,China;National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州省病原生物学特色重点实验室,贵阳550025 [2]中国疾病预防控制中心传染病预防控制所,传染病溯源预警与智能决策全国重点实验室,北京102206 [3]中国疾病预防控制中心病毒病预防控制所,传染病溯源预警与智能决策全国重点实验室,北京100052

出　　处：《中国人兽共患病学报》2024年第5期435-441,共7页Chinese Journal of Zoonoses

基　　金：国家自然科学基金青年科学基金项目(No.31800004);中国疾病预防控制中心传染病预防控制所自主课题(No.2021ZZKT003)。

摘　　要：目的从食品销售环境样本中分离李斯特菌噬菌体,并对分离获得的噬菌体进行电镜形态观察、宿主谱及生物学特征分析。方法采用双层琼脂平板法和点滴法,以分离的英诺克李斯特菌Lin08作为宿主菌,成功分离并鉴定出一株烈性噬菌体LMLPA5,使用透射电镜观察噬菌体形态,并测定噬菌体的宿主谱、最佳感染复数(MOI)、一步生长曲线以及理化稳定性,同时探索噬菌体在4℃、-20℃及-80℃下的保存效果。结果分离获得的LMLPA5噬菌体属于肌尾噬菌体科,其形成的噬菌斑清晰透明,周围无晕环。该噬菌体为广谱的李斯特菌烈性噬菌体,能裂解多个李斯特菌种及单增李斯特菌多个血清型菌株。LMLPA5的最佳MOI为0.1,潜伏期为10 min,平均裂解量为95.2 PFU/cell。LMLPA5对高温较敏感,在70℃暴露1 h即完全失活,而在4℃~40℃作用32 h以上噬菌体仍能保持稳定。在pH为4~10 LMLPA5的活性不受影响,经紫外线照射60 min后噬菌体被完全灭活。LMLPA5对氯仿不敏感,为无囊膜型噬菌体。噬菌体在4℃及-80℃的保存效果较好,可稳定保存8个月以上。结论本研究获得的1株广谱李斯特菌肌尾噬菌体LMLPA5具有较强的裂解能力、抵抗力和稳定性,为进一步的应用提供了基础。The aim of our research was to obtain Listeria bacteriophages from food and related environments,and conducted the analysis of the electron microscopic morphology,host range specificity,and biological characteristics of the purified phages.The double-layer agar method and the spot test were employed for the isolation and identification of a virulent Listeria phage named LMLPA5,with the isolated strain Listeria innocua Lin08 as the host.Phage morphology was observed by transmission electron microscope.The biological characteristics of the phage were assessed by determining their host range,optimal multiplicity of infection(MOI),one-step growth curve,and physicochemical stability.Additionally,the preservation efficacy of the phage at 4℃,-20℃,and-80℃was explored.The phage LMLPA5 belongs to the family Myoviridae based on morphology,exhibiting clear and transparent plaques without halo surrounded.Strains of several Listeria species and different serotypes strains of Listeria monocytogenes were susceptible to lysis by LMLPA5,indicating its broad-spectrum activity against Listeria monocytogenes.Optimal MOIs and single-step growth curve analyses revealed optimal MOIs of 0.1 and latent period of 10 minutes for LMLPA5,with average burst size at 95.2 PFU/cell.LMLPA5 was sensitive to high temperatures,and completely inactivated after exposure to 70℃for 1 h,while the phage remained stable for over 32 hours ranging from 4℃to 40℃.Within the pH range of 4 to 10,phage titer remained stable and completely inactivated until 60 minutes of ultraviolet exposure.LMLPA5 displayed insensitivity to chloroform,confirming its non-enveloped phage morphology.The phages remained stable for over 8 months when store at 4℃and-80℃.The biological characteristics and lysis capacity of phage LMLPA5 were elucidated in this study,which provide the basis for further application.

关 键 词：李斯特菌噬菌体 裂性噬菌体 生物学特性 

分 类 号：R383.3[医药卫生—医学寄生虫学]