摘要
目的以表达绿色荧光蛋白(GFP)报告基因的重组腺病毒(rAdvGFP)为模式病毒,进行气溶胶的主动发生实验和模拟剧烈吹打、振荡混匀等操作,利用安德森采样器进行气溶胶的采集,以建立简单易行的病毒气溶胶采集检测体系。方法用气溶胶发生器模拟模式病毒的气溶胶主动发生,分别在距离发生器管口0、10和20cm处放置采样器及培养皿,在气溶胶发生30s时开始采集,共采集5min。在模拟吹打操作中,将10ml病毒液置于50ml离心管中,用微量移液器反复吸取吹打病毒液;在模拟振荡操作中,将10ml病毒液置于50ml离心管中盖紧,以振荡器最大速度涡旋振荡1min,立即打开管盖采集2min,然后盖紧再重复振荡30s后开盖继续采集。两种模拟操作均在距离管口0和10cm处放置采样器及培养皿,各采集5min。收集各采样器及培养皿中的液体,利用阳离子聚合物聚凝胺浓缩后用PCR方法检测病毒核酸,并接种人胚肾293细胞进行病毒的培养,在荧光显微镜下观察GFP蛋白的表达。结果模拟气溶胶主动发生时,将置于距发生器管口0、10和20cm的采样器和培养皿中的采集液接种细胞均可观察到绿色荧光,并可检测到病毒核酸。模拟剧烈吹打时,置于0cm处的培养皿内可检测到病毒,而置于0cm、10cm处采样器和10cm处培养皿中均未检测到病毒。在剧烈振荡模拟试验中,在距离管口0cm的采样器和培养皿中均可检测到病毒,在置于10cm处的采样器中未检测到病毒,在置于10cm处的培养皿中可检测到病毒。结论利用rAdvGFP模式病毒和安德森采样器初步建立了病毒学实验气溶胶收集检测体系,对模拟危险操作感染性材料所产生的气溶胶做出危险评估,为制定病毒学实验操作规范提供了依据。
Objective To establish viral aerosol collection system and to evaluate the hazards of dangerous operations, Andersen microbial air sampler was used and two most frequently operations i.e. pipetting or vortex liquid containing viruses were mimicked, by using a recombinant adenovirus expressing green fluorescent protein （GFP） as a model.Methods The recombinant adenovirus expressed green fluorescence protein （rAdvGFP） model viruses were aerosolized using the aerosol generator and sampled by the andersen microbial air sampler. The captured chambers of the sampler were put on the position with 0 cm, 10 cm and 20 cm horizontal distance to the aerosol generator. Viral particles were collected for total 5 minutes （mins） after the artificially produced aerosols for 30 seconds （sec）.When mimicking the dangerous operations, 10 ml culture medium contained viruses in 50 ml screw tube was used and chambers of the sampler were put on the position with 0 cm and 10 cm horizontal distance to the tube. The viruses were pipetted violently by using pipettes and aerosol was sampled for 5 mins. When mimicking viruses vibration, the tube was vortexed at the highest speed on the vortex for 1 min and sampled for 2 mins after opening the cap of tube, then snapped the lid on before vortexed again for 30 secs and sampled total for 5rains. The medium in the tube was collected, enriched and cell-culture-propagated on HEK 293 cells. Nucleic acids were tested by using PCR method. Viral propagation was checked by observed GFP expression using fluorescent microscope.Results The dishes and sampler of 0 cm, 10 cm and 20 cm group showed green fluorescence after the artificially produced aerosols When mimicking pipetting violently, the 0 cm dishes recovered model viruses, but no model viruses were detected in 10 cm dishes and 0 cm and 10 cm sampler. When mimicking vibration, model viruses were recovered from dishes and samplers at 0 cm group and 10 cm dishes, while no viruses were captured from dishes and samplers at 10 cm group.Conclusions By using rAdvGFP model viruses, a system focus on viral aerosol collection and detection was established. The system facilitate to assay the risk of dangerous operations on aerosol generation and to make the standard operating procedures in biosafety cabinet based on such evaluations.
出处
《中国医药生物技术》
CSCD
2010年第5期342-347,共6页
Chinese Medicinal Biotechnology
基金
国家卫生行业科研专项(200802021)
