本文選題：噬菌體 + 分離及特性��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年博士論文

【摘要】： 自20世紀70年代以來,在世界范圍內(nèi)發(fā)現(xiàn)和確認的新發(fā)傳染病已多達40余種,在這些傳染病中約50%可通過空氣傳播,而且?guī)缀趺磕曛辽儆幸环N新的傳染病被發(fā)現(xiàn),它們對人類健康、社會經(jīng)濟發(fā)展造成了嚴重的威脅。如2003年爆發(fā)的SARS,僅9個月,全世界30個國家有8439人感染,812人死亡;2004年起發(fā)生的高致病性禽流感;以及去年爆發(fā)的甲型H1N1流感,均造成了人員和財產(chǎn)的重大損失。 防護裝備和設(shè)施在隔離傳染源、阻斷微生物氣溶膠的傳播途徑和保護易感人群三方面均發(fā)揮著重要作用,在面對大范圍未知生物恐怖襲擊事件、自然突發(fā)事件時,是阻止疫病擴散最快最有效的方式之一,可起到立竿見影的效果。盡管如此,有些防護裝備和設(shè)施仍然存在生物學(xué)防護效果評價方法或標準不完善甚至缺乏的情況。 本研究旨在建立和完善生物防護裝備和設(shè)施對呼吸道病毒替代病毒氣溶膠防護效果評價技術(shù)與平臺,提升對生物防護裝備和設(shè)施進行模擬病毒氣溶膠防護效果評價的檢測能力;開展對生物防護口罩、生物防護服、正壓醫(yī)用防護頭罩、負壓隔離轉(zhuǎn)運艙、負壓救護車排風(fēng)凈化裝置、Ⅱ級生物安全柜和高等級生物安全實驗室排風(fēng)系統(tǒng)等生物防護裝備和設(shè)施的安全性能評價。 內(nèi)容: 1.從污水中分離大腸桿菌和粘質(zhì)沙雷氏菌噬菌體并對其生物學(xué)特點進行系統(tǒng)研究; 2.研究模式病毒耐受發(fā)生和采樣壓力特點; 3.建立和完善生物防護裝備和設(shè)施評價技術(shù)與平臺并對多種防護裝備和設(shè)施進行病毒氣溶膠防護效果測試評價; 方法: 1.采用四步法從污水中分離大腸桿菌和粘質(zhì)沙雷氏菌噬菌體,通過單層平板噬菌斑和雙層平板噬菌斑實驗篩選烈性噬菌體,挑取單個典型噬菌斑進行噬菌體的培養(yǎng)增殖及純化,電鏡觀察噬菌體的形態(tài),對噬菌體最佳感染復(fù)數(shù)、一步生長曲線特點、紫外線滅活特點、噬菌體寬噬性進行了研究,手工提取噬菌體核酸并進行電泳分析,對噬菌體結(jié)構(gòu)蛋白進行了聚丙烯酰胺凝膠電泳分析; 2.在密閉柜內(nèi)使用玻璃發(fā)生器Devilbiss 40發(fā)生模式病毒氣溶膠,發(fā)生液分別選用肉湯、SM液和PBS,TSI-3321氣溶膠粒子分析儀測量空氣中模式病毒氣溶膠粒子譜,分別于發(fā)生前后取少量發(fā)生液,雙層瓊脂平板法測定噬菌體滴度,使用統(tǒng)計軟件對結(jié)果進行分析; 3.使用全玻璃液體沖擊式采樣器(AGI-10)對模式病毒進行耐沖擊實驗,采樣液選用蒸餾水(DW)、磷酸鹽緩沖液(PBS)和SM液,每種采樣液又分為加橄欖油組和不加橄欖油組,在以7L/min的氣流沖擊30min后測定采樣液中噬菌體滴度和終末采樣液體積,采用校正存活率評價噬菌體耐沖擊性,使用統(tǒng)計軟件對結(jié)果進行分析; 4.發(fā)生模式病毒氣溶膠,空氣微生物采樣器采集防護裝備和設(shè)施氣溶膠暴露區(qū)和被保護區(qū)空氣中模式病毒粒子,采用防護效率評價防護裝備和設(shè)施對病毒氣溶膠的防護效果。 結(jié)果: 1.成功分離出粘質(zhì)沙雷氏菌烈性噬菌體2株(SM701、SM702),噬菌體SM701和SM702在雙層平板上分別培養(yǎng)6h和8h后可出噬菌斑,前者噬菌斑形態(tài)為圓形,直徑1mm左右(培養(yǎng)12h),噬菌斑透亮度較高;后者噬菌斑圓形,直徑2mm～3mm左右(培養(yǎng)12h),透亮度較前者低。兩株噬菌體形態(tài)極為相似,均有一個正多面體立體對稱的頭部,頭徑約64nm,無囊膜,有一長尾,無收縮尾鞘,尾長約143nm。兩者最佳感染復(fù)數(shù)均為10。SM701感染宿主菌的潛伏期約為30min,爆發(fā)時間約為100min,裂解量約為63;SM702感染宿主菌的潛伏期約為40min,爆發(fā)時間約為90min,裂解量約為5。SM701和SM702在紫外線(光強221μW/cm2)下分別暴露14min和16min全部失活。噬菌體SM701和SM702核酸類型為dsDNA,衣殼和尾至少分別由14和16個不同大小蛋白構(gòu)成,兩者之間相似大小條帶8條。 成功分離出大腸桿菌(285)烈性噬菌體2株(EcP1、EcP2), EcP1噬菌斑直徑3mm～5mm(培養(yǎng)12h),逆光觀察噬菌斑呈全透明狀,該噬菌體有一個長多面體立體對稱的頭部,頭長徑(L)約47nm,頭橫徑(W)約35nm,L/W=1.34,無囊膜,有一短尾,尾長約20nm;EcP2噬菌斑直徑約1mm(培養(yǎng)12h),逆光觀察噬菌斑呈全透明狀,該噬菌體有一個長多面體立體對稱的頭部,頭長徑(L)約89nm,頭橫徑(W)約54nm,L/W=1.65,無囊膜,有一長尾,有尾鞘,尾長約81nm。EcP2可噬大腸桿菌(8099)形成直徑約1mm的圓形噬菌斑。EcP1和EcP2最佳感染復(fù)數(shù)分別為10和0.1,在紫外燈(光強221μW/cm2)下暴露8min和4min可全部失活。EcP1和EcP2核酸類型為dsDNA,衣殼和尾至少分別由12和16個不同大小蛋白構(gòu)成。 2.在19.5min內(nèi),發(fā)生器Devilbiss 40對懸液中噬菌體活性影響小,影響隨模式病毒和發(fā)生液的不同而不同;不同發(fā)生液之間發(fā)生前噬菌斑數(shù)差別無統(tǒng)計學(xué)意義(P0.05),發(fā)生后19.5min三種不同發(fā)生液之間噬菌斑數(shù)差異具有統(tǒng)計學(xué)意義(P0.01),均值結(jié)果為肉湯SM液PBS,說明相同滴度的噬菌體,使用三種不同的發(fā)生液,發(fā)生一定時間后,噬菌體活性之間差別顯著,使用肉湯作為發(fā)生液對噬菌體的保護作用最好,不同模式病毒氣溶膠粒子數(shù)量中值直徑在0.7μm到0.8μm之間。 3.同一種噬菌體在不同采樣液中,耐沖擊性不同,以SM液作為采樣液時,噬菌體存活率較高,SM液中是否加入橄欖油對噬菌體的耐沖擊性沒有影響。噬菌體SM701、SM702、PhiX174、EcP1和F2在SM液中經(jīng)7L/min的氣流沖擊60min后,校正存活率分別為79%、77%、86%、50%和71%左右。在短時間(10min)內(nèi),噬菌體SM701、SM702、PhiX174和f2存活率無差別。 4.國內(nèi)市售醫(yī)用防護口罩質(zhì)量良莠不齊,隨檢測產(chǎn)品的不同病毒學(xué)檢測達標率為0、20%、60%和100%;正壓醫(yī)用防護頭罩對病毒氣溶膠的防護效率大于99.98%;從醫(yī)用防護服測試結(jié)果來看,我國目前沒有能夠達到國際標準ISO16604的市售防護服;固定式和充氣式負壓隔離轉(zhuǎn)運艙病毒氣溶膠隔離效率均大于99.999%;負壓救護車排風(fēng)負壓凈化裝置病毒氣溶膠過濾效率大于99.99%;三級生物安全實驗室中9塊被檢測HEPA中,有1塊HEPA存在模式病毒氣溶膠漏;生物安全柜檢測結(jié)果表明,不同指示微生物在人員、產(chǎn)品和交叉污染保護實驗中結(jié)果無明顯差異,但高效空氣粒子過濾器生物學(xué)檢漏結(jié)果隨指示微生物是細菌還是病毒而不同。 結(jié)論: 1.四步法是一種有效的噬菌體污水分離方法; 2.噬菌體SM701和SM702屬于長尾噬菌體科噬菌體,噬菌體EcP1和EcP2分別屬于短尾噬菌體科噬菌體和肌尾噬菌體科噬菌體; 3.成功分離的噬菌體SM702易于培養(yǎng)計數(shù)且對發(fā)生和采樣沖擊壓力耐受,是理想的空氣微生物學(xué)研究示蹤微生物;營養(yǎng)肉湯是理想的氣溶膠發(fā)生液;SM液是理想的液體沖擊式采樣器采樣液; 4.噬菌體SM702可模擬病毒氣溶膠對多種生物防護裝備和設(shè)施病毒氣溶膠防護效果進行評價; 5.成功建立和完善了生物防護裝備和設(shè)施病毒氣溶膠防護效果評價技術(shù)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R82

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