模式病毒（噬菌体）分离、特性及在防护装备和设施评价中的应用研究

发布时间：2018-05-29 11:32

本文选题：噬菌体 + 分离及特性　；参考：《中国人民解放军军事医学科学院》2010年博士论文

【摘要】： 自20世纪70年代以来,在世界范围内发现和确认的新发传染病已多达40余种,在这些传染病中约50%可通过空气传播,而且几乎每年至少有一种新的传染病被发现,它们对人类健康、社会经济发展造成了严重的威胁。如2003年爆发的SARS,仅9个月,全世界30个国家有8439人感染,812人死亡;2004年起发生的高致病性禽流感;以及去年爆发的甲型H1N1流感,均造成了人员和财产的重大损失。防护装备和设施在隔离传染源、阻断微生物气溶胶的传播途径和保护易感人群三方面均发挥着重要作用,在面对大范围未知生物恐怖袭击事件、自然突发事件时,是阻止疫病扩散最快最有效的方式之一,可起到立竿见影的效果。尽管如此,有些防护装备和设施仍然存在生物学防护效果评价方法或标准不完善甚至缺乏的情况。本研究旨在建立和完善生物防护装备和设施对呼吸道病毒替代病毒气溶胶防护效果评价技术与平台,提升对生物防护装备和设施进行模拟病毒气溶胶防护效果评价的检测能力;开展对生物防护口罩、生物防护服、正压医用防护头罩、负压隔离转运舱、负压救护车排风净化装置、Ⅱ级生物安全柜和高等级生物安全实验室排风系统等生物防护装备和设施的安全性能评价。内容: 1.从污水中分离大肠杆菌和粘质沙雷氏菌噬菌体并对其生物学特点进行系统研究; 2.研究模式病毒耐受发生和采样压力特点; 3.建立和完善生物防护装备和设施评价技术与平台并对多种防护装备和设施进行病毒气溶胶防护效果测试评价; 方法: 1.采用四步法从污水中分离大肠杆菌和粘质沙雷氏菌噬菌体,通过单层平板噬菌斑和双层平板噬菌斑实验筛选烈性噬菌体,挑取单个典型噬菌斑进行噬菌体的培养增殖及纯化,电镜观察噬菌体的形态,对噬菌体最佳感染复数、一步生长曲线特点、紫外线灭活特点、噬菌体宽噬性进行了研究,手工提取噬菌体核酸并进行电泳分析,对噬菌体结构蛋白进行了聚丙烯酰胺凝胶电泳分析; 2.在密闭柜内使用玻璃发生器Devilbiss 40发生模式病毒气溶胶,发生液分别选用肉汤、SM液和PBS,TSI-3321气溶胶粒子分析仪测量空气中模式病毒气溶胶粒子谱,分别于发生前后取少量发生液,双层琼脂平板法测定噬菌体滴度,使用统计软件对结果进行分析; 3.使用全玻璃液体冲击式采样器(AGI-10)对模式病毒进行耐冲击实验,采样液选用蒸馏水(DW)、磷酸盐缓冲液(PBS)和SM液,每种采样液又分为加橄榄油组和不加橄榄油组,在以7L/min的气流冲击30min后测定采样液中噬菌体滴度和终末采样液体积,采用校正存活率评价噬菌体耐冲击性,使用统计软件对结果进行分析; 4.发生模式病毒气溶胶,空气微生物采样器采集防护装备和设施气溶胶暴露区和被保护区空气中模式病毒粒子,采用防护效率评价防护装备和设施对病毒气溶胶的防护效果。结果: 1.成功分离出粘质沙雷氏菌烈性噬菌体2株(SM701、SM702),噬菌体SM701和SM702在双层平板上分别培养6h和8h后可出噬菌斑,前者噬菌斑形态为圆形,直径1mm左右(培养12h),噬菌斑透亮度较高;后者噬菌斑圆形,直径2mm～3mm左右(培养12h),透亮度较前者低。两株噬菌体形态极为相似,均有一个正多面体立体对称的头部,头径约64nm,无囊膜,有一长尾,无收缩尾鞘,尾长约143nm。两者最佳感染复数均为10。SM701感染宿主菌的潜伏期约为30min,爆发时间约为100min,裂解量约为63;SM702感染宿主菌的潜伏期约为40min,爆发时间约为90min,裂解量约为5。SM701和SM702在紫外线(光强221μW/cm2)下分别暴露14min和16min全部失活。噬菌体SM701和SM702核酸类型为dsDNA,衣壳和尾至少分别由14和16个不同大小蛋白构成,两者之间相似大小条带8条。成功分离出大肠杆菌(285)烈性噬菌体2株(EcP1、EcP2), EcP1噬菌斑直径3mm～5mm(培养12h),逆光观察噬菌斑呈全透明状,该噬菌体有一个长多面体立体对称的头部,头长径(L)约47nm,头横径(W)约35nm,L/W=1.34,无囊膜,有一短尾,尾长约20nm;EcP2噬菌斑直径约1mm(培养12h),逆光观察噬菌斑呈全透明状,该噬菌体有一个长多面体立体对称的头部,头长径(L)约89nm,头横径(W)约54nm,L/W=1.65,无囊膜,有一长尾,有尾鞘,尾长约81nm。EcP2可噬大肠杆菌(8099)形成直径约1mm的圆形噬菌斑。EcP1和EcP2最佳感染复数分别为10和0.1,在紫外灯(光强221μW/cm2)下暴露8min和4min可全部失活。EcP1和EcP2核酸类型为dsDNA,衣壳和尾至少分别由12和16个不同大小蛋白构成。 2.在19.5min内,发生器Devilbiss 40对悬液中噬菌体活性影响小,影响随模式病毒和发生液的不同而不同;不同发生液之间发生前噬菌斑数差别无统计学意义(P0.05),发生后19.5min三种不同发生液之间噬菌斑数差异具有统计学意义(P0.01),均值结果为肉汤SM液PBS,说明相同滴度的噬菌体,使用三种不同的发生液,发生一定时间后,噬菌体活性之间差别显著,使用肉汤作为发生液对噬菌体的保护作用最好,不同模式病毒气溶胶粒子数量中值直径在0.7μm到0.8μm之间。 3.同一种噬菌体在不同采样液中,耐冲击性不同,以SM液作为采样液时,噬菌体存活率较高,SM液中是否加入橄榄油对噬菌体的耐冲击性没有影响。噬菌体SM701、SM702、PhiX174、EcP1和F2在SM液中经7L/min的气流冲击60min后,校正存活率分别为79%、77%、86%、50%和71%左右。在短时间(10min)内,噬菌体SM701、SM702、PhiX174和f2存活率无差别。 4.国内市售医用防护口罩质量良莠不齐,随检测产品的不同病毒学检测达标率为0、20%、60%和100%;正压医用防护头罩对病毒气溶胶的防护效率大于99.98%;从医用防护服测试结果来看,我国目前没有能够达到国际标准ISO16604的市售防护服;固定式和充气式负压隔离转运舱病毒气溶胶隔离效率均大于99.999%;负压救护车排风负压净化装置病毒气溶胶过滤效率大于99.99%;三级生物安全实验室中9块被检测HEPA中,有1块HEPA存在模式病毒气溶胶漏;生物安全柜检测结果表明,不同指示微生物在人员、产品和交叉污染保护实验中结果无明显差异,但高效空气粒子过滤器生物学检漏结果随指示微生物是细菌还是病毒而不同。结论: 1.四步法是一种有效的噬菌体污水分离方法; 2.噬菌体SM701和SM702属于长尾噬菌体科噬菌体,噬菌体EcP1和EcP2分别属于短尾噬菌体科噬菌体和肌尾噬菌体科噬菌体; 3.成功分离的噬菌体SM702易于培养计数且对发生和采样冲击压力耐受,是理想的空气微生物学研究示踪微生物;营养肉汤是理想的气溶胶发生液;SM液是理想的液体冲击式采样器采样液; 4.噬菌体SM702可模拟病毒气溶胶对多种生物防护装备和设施病毒气溶胶防护效果进行评价; 5.成功建立和完善了生物防护装备和设施病毒气溶胶防护效果评价技术方法。
[Abstract]:Since 1970s, more than 40 new infectious diseases have been found and identified worldwide. About 50% of these infectious diseases are transmitted through air, and at least one new infectious disease is found in almost every year. They have made a serious threat to human health and social and economic development. For example, only 9 of the outbreak of SARS in 2003. In 30 countries, 8439 people were infected and 812 people died in the world. The high pathogenic avian influenza in 2004, and the outbreak of H1N1 influenza a year, all caused significant loss of personnel and property.
Protective equipment and facilities play an important role in isolating the source of infection, blocking the transmission of microbial aerosol and protecting the susceptible population in three aspects. In the face of large unknown biological terrorist attacks and natural emergencies, it is one of the fastest and most effective ways to prevent the spread of the epidemic disease. In this regard, some protective equipment and facilities still exist the evaluation method or standard of biological protection effect is imperfect or even lacking.
The purpose of this study is to establish and improve the evaluation technology and platform of the biological protective equipment and facilities for the protection effect of the respiratory virus replacement virus aerosol, and to improve the detection ability of the evaluation of the protective effect of the biological protective equipment and facilities for the simulation of the protective effect of the virus aerosol, and to carry out the biological protective cover, biological protective clothing and positive pressure medical protective cover. The safety performance evaluation of biological protective equipment and facilities such as pressure isolation transport cabin, negative pressure ambulance exhaust cleaning device, class II biosafety cabinet and high grade biological safety laboratory exhaust system.
Content:
1. isolation of Escherichia coli and Serratia marcescens phage from sewage and its biological characteristics.
2. research pattern tolerance and sampling pressure.
3. to establish and improve the evaluation technology and platform of biological protective equipment and facilities, and to test and evaluate the protective effect of virus aerosol on a variety of protective equipment and facilities.
Method:
1. the four step method was used to isolate the bacteriophages of Escherichia coli and Serratia mucilagus from sewage. The bacteriophages were screened by single plate phage plaque and double plate phage plaque experiment, and single typical phage plaque was selected for the proliferation and purification of phage, the morphology of phage was observed by electron microscope, the number of best phage infection complex number and one step growth were observed. The characteristics of the curves, the characteristics of ultraviolet inactivation, the wide phage phage of phage were studied. The phage nucleic acid was extracted by hand and analyzed by electrophoresis. The phage structure protein was analyzed by polyacrylamide gel electrophoresis.
2. using the glass generator Devilbiss 40 in the closed cabinet, the model virus aerosol was used, and the broth, the SM solution and the PBS, and the TSI-3321 aerosol particle analyzer were used to measure the aerosol particle spectrum of the pattern virus in the air. A small amount of fluid was taken before and after the occurrence, and the titer of the phage was measured by the double layer agar plate method, and the statistical software was used. The results are analyzed.
3. the whole glass liquid impact sampler (AGI-10) was used to test the impact resistance of the model virus. The sample solution was distilled water (DW), phosphate buffer solution (PBS) and SM solution. Each sample was divided into olive oil group and non olive oil group. The phage titer and final sampling liquid in the sample was determined after the impact of 7L/min on the air flow impact 30min. The corrected survival rate was used to evaluate the impact resistance of phages, and the results were analyzed by statistical software.
4. model virus aerosol, air microorganism sampler collect protective equipment and facilities aerosol exposure area and protected area air model virus particles, and use protective efficiency to evaluate protective effect of protective equipment and facilities on virus aerosol.
Result:
1. successfully isolated 2 strains of phage phage (SM701, SM702), phage SM701 and SM702, the phage SM701 and SM702 were separately cultured for 6h and 8h plaque. The former phage plaque was round, the diameter of the phage was around 1mm (culture 12h), and the phage plaque was high in brightness; the latter was round, the diameter was 2mm to 3mm (12h), and the brightness was better than before. The two phage forms are very similar. There is a positive polyhedron with a stereoscopic head with a head diameter of about 64nm, no capsule, a long tail, no contractile tail sheath, and a tail length about 143nm. and 10.SM701 infection. The incubation period of the host bacteria is about 30min, the outbreak time is about 100min, and the amount of lysis is about 63; SM702 infected host bacteria. The incubation period is about 40min, and the outbreak time is about 90min. The lysis amount is about 5.SM701 and SM702 in the ultraviolet (light intensity 221) W/cm2, respectively. 14min and 16min are completely deactivated. The phage SM701 and SM702 nucleic acid types are dsDNA, and the capsid and tail are composed of at least 14 and 16 different sizes of proteins respectively, and there are 8 similar bands between them.
The bacteriophage (EcP1, EcP2) of Escherichia coli (285) was successfully isolated, and the phage plaque in EcP1 was 3mm to 5mm (12h). The phage was fully transparent. The phage had a long polyhedral and stereoscopic head, the head length (L) about 47nm, the head transverse diameter (W) of 35nM, L/W=1.34, and the tail length about 20nm; the tail length was about 20nm; the plaque was straight. About 1mm (culture 12h), the phage was fully transparent. The phage had a long polyhedral and stereoscopic head, the head length (L) was about 89nm, and the head transverse diameter (W) was about 54nm, L/W=1.65, and no capsule. There was a long tail, a tail sheath, and the tail length was about 81nm.EcP2 of Escherichia coli (8099) to form a circular plaque.EcP1 and EcP2 optimal infection with a diameter of about 1mm. The plural numbers were 10 and 0.1 respectively. The exposure of 8min and 4min to the ultraviolet light (light intensity 221 W/cm2) could completely deactivate.EcP1 and EcP2 nucleic acid type dsDNA, and the capsid and tail were composed of at least 12 and 16 different proteins respectively.
2. in 19.5min, the effect of the generator Devilbiss 40 on the phage activity in the suspension was small, and the effect was different with the pattern virus and the occurring fluid. There was no significant difference between the number of phage spots before the occurrence of different occurring fluids (P0.05). The difference of the number of phage spots among the three different 19.5min fluids after the occurrence was statistically significant (P0.01). The fruit is the broth SM liquid PBS, indicating the phage with the same titer. Using three different kinds of occurrence liquid, after a certain time, the bacteriophage activity has a significant difference. The use of broth as the occurrence solution is the best for the bacteriophage protection. The diameter of the different model virus aerosol particles is between 0.7 and 0.8 mu m.
3. the same phage has different impact resistance in different samples. When SM solution is used as sampling solution, the survival rate of phage is higher. The addition of olive oil in SM solution has no effect on the impact resistance of phage. The corrected survival rate is 79%, 77%, 86%, respectively, after SM701, SM702, PhiX174, EcP1 and F2 in SM liquid. About 50% and 71%, there was no difference in the survival rate of phage SM701, SM702, PhiX174 and F2 within a short time (10min).
4. the quality of medical protective masks for sale in domestic market is different, with the detection rate of 0,20%, 60% and 100% with different virology detection products, the protection efficiency of positive pressure medical protective cover is more than 99.98%. From the result of medical protective clothing test, we have not reached the international standard ISO16604. The isolation efficiency of the aerosols was greater than 99.999% in the fixed and inflatable negative pressure isolation transport tanks, and the filtration efficiency of the virus aerosol was more than 99.99% in the negative pressure ambulance negative pressure purification device, and 1 HEPA in the three biosafety laboratory was detected in HEPA, and the results of biological safety cabinet showed that different fingers were different. There were no significant differences in the results of microbes in personnel, products and cross contamination protection experiments, but the biological leak detection results of the high efficiency air particle filter vary with the bacteria or virus.
Conclusion:
1. the four step method is an effective method for bacteriophage wastewater separation.
2. phage SM701 and SM702 belong to the long tail phage family bacteriophages. Phage EcP1 and EcP2 belong to the bacteriophages of the short tail phage family and bacteriophages of muscle tail bacteriophages.
3. the successful isolated phage SM702 is easy to count and to be tolerant to the occurrence and sampling of impact pressure. It is an ideal trace microorganism for the study of air microbiology, and the nutritive broth is an ideal aerosol generation liquid, and the SM liquid is the ideal liquid impact sampler sampling solution.
4. phage SM702 can simulate the effect of virus aerosol on the protective effect of various biological protective equipment and facilities.
5. we have successfully established and perfected the technical methods for evaluating the protective effect of aerosol aerosol in biological protective equipment and facilities.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R82

【参考文献】