空气和水环境中病毒的富集、检测与净化

【作者】 陈军红；

【导师】 周志军；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 硕士

【摘要】 空气、水中的一些致病病毒是疾病传播的一个重要途径,如SARS病毒、甲型肝炎病毒等。这些病毒在空气和水中的浓度极低,而且需要特定的方法检测,而大气、水的通常采样方法都很难做到对病毒的富集与检测。病毒不同于细菌、真菌等微生物,不能通过常规培养基培养观察。病毒的检测通常需借助特殊的仪器和方法,操作复杂、要求严格,其目的主要是检测某种特殊病毒是否存在及含量。对于空气和水环境中的病毒,可用膜过滤装置进行截留,用于环境净化或富集后用于检测。本课题采用孔径很小的超滤膜作为空气、水采样的介质,可以高效地富集环境中的极微量病毒,富集的病毒可用细胞培养法、PCR法等检测。主要以自然环境中容易分离得到的噬菌体为模型病毒,以双层琼脂法培养的噬菌斑为病毒存在的直观指示,通过测定空气和水环境中模型病毒经膜过滤装置过滤前后的浓度变化来评价膜装置富集病毒的效果。这种方法简易、高效、灵敏、可靠,为研制和丌发各种病毒采样富集装置或环境病毒净化装置提供了合适的测试技术平台。另外,本课题还初步研制开发了用于空气病毒净化的膜装置,并对其截留空气中微量病毒的性能进行了测试。在病毒的采样、富集与检测以及空气病毒净化方面,大肠杆菌噬菌体是一种很好的模型病毒,其制备包括从污水中分离噬菌体、纯化噬菌体及增殖制备高效价噬菌体。实验中采用了各种不同的超滤膜来截留空气、水中的大肠杆菌噬菌体和甲型肝炎减毒活疫苗,对噬菌体的检测用双层琼脂法,对甲肝疫苗的检测采用酶联免疫试剂盒的方法。实验结果表明,膜过滤法截留噬菌体并用双层琼脂法检测是一种简单可行地模拟环境病毒富集与检测的方法。超滤膜的截留率可达到100%,将病毒富集后用双层琼脂法检测,检测限约10PFU/ml。以此方法测试空气病毒净化器的性能表明,初步研制的膜法空气病毒净化装置能够完全去除空气中的微量病毒,有望应用在医院传染病房、生物安全实验室的空气循环系统。更多还原

【Abstract】 Environmental viruses (airborne or waterborne) such as SARS and HAV are basic origin of epidemic diseases. Conventional sampling methods in air and water are difficult to detect these viruses, which are of very low concentration in outside environment and need specific detecting methods. Viruses, different to bacteria and fungi, can’ t live on general culture media, and their detection lies on specific method and complicated apparatus. It’ s suitable to enrich and detect airborne or waterborne viruses or to remove them using membrane-intercepting method.This thesis is to enrich efficiently small amount of viruses in air and water using ultra-filtration membrane as sampling media. Cell culturing or PCR method can be used to detect the enriching viruses. In our experiments, phage is usually used as model virus to simulate membrane enriching of real environmental viruses. The enriching effect of membrane is evaluated by detecting the concentration change of virus across the membrane. Double layer agar method to detect phage is used, which is intuitionistic to display plaque. The process is simple, efficient and sensitive so as to supply a good technical flat of developing virus-enriching or virus-cleaning equipment. Membrane equipment of cleaning airborne virus and its performance is also researched in this thesis.The preparation of model virus (phage) includes separating phage from wasting water, purifying it and multiplication. Various ultra-filtration membranes are used to intercept and capture phage and HAV in air & water. HAV is detected by EIA-kit method. The results indicate that utilizing membrane filtration to intercept and capture phage and then using double layer agar method to detect it are simple and practical for the simulation of sampling and detecting environmental viruses. The rate of cut-off isup to 100% and the detecting limit is about 10PFU/ml. The primarily developed airborne virus cleaning equipment can remove small amount of viruses in air completely. This equipment has great prospect applying in air circulating system of infecting sickroom and bio-safety laboratory.更多还原