SARS病人病房空气污染和SARS病毒空气分离株的基础研究及其相关病毒数据库的建立
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摘要
SARS-CoV是一种新型的单链RNA人畜共患病病毒,该病毒主要通过近距离气溶胶和亲密接触传播,在家庭和医院有显著的聚集现象。空气传播感染受多种因素制约,其中最重要的是SARS-CoV在气溶胶状态下的存活力,以及环境的温度、湿度、紫外线和空气中各种化学污染物质。
本研究旨在通过对SARS病人定点收治医院(小汤山医院、309医院)室内空气SARS-CoV污染的研究,为更好地控制SARS-CoV的空气传播感染提供实验依据。实验采用FA-Ⅱ型空气微生物采样器在医院病房区、病房阳台、内走廊、排风扇和护士站五个地点持续采样,之后进行空气样本的洗脱、Veto-E6细胞培养、RT-PCR及序列测定。其结果病房区、病房阳台、内走廊、排风扇和清洁区五个地点均有SARS-CoV RNA的检出,其中以小汤山医院排气口下风向的阳性率最高(58.3%),护士站相对较低(25.0%);从309医院一名确诊为SARS病人病房内的空气样本中分离出一株活性SARS-CoV(称之为LK309株),并稳定传代;与恢复期SARS病人血清免疫荧光染色阳性,RT-PCR扩增产物的测序结果显示LK309株与已知SARS-CoY的同源性在98%以上。相关数据表明SARS病人定点收治医院空气中SARS-CoV的污染比较严重,急性后期的病人仍然从呼吸道大量排毒;但在病房室内外的空气中几乎没有检测到活的SARS-CoV,初步评估认为,SARS病人病房采取通风和消毒的措施对降低室内污染程度是非常有益的;而空气消毒和环境因素对SARS病毒的存活有较大的影响,故SARS病人病房排出的空气对周边环境造成的危害很小。
针对SARS-CoY空气分离株LK309,根据引物步移法的原则,通过覆盖全基因
SARS-CoV is a kind of novel single RNA virus which can arose zoonosis. This kind of virus can spread quickly via short-haul airborne infection and hand-in-hand infection. In hospital and at home SARS-CoV takes on prominent assembly phenomena. Admittedly airborne infection can be interfered by multi-fold factors, the most important factor is livability of SARS-CoV in the aerosol state, and the others include temperature and humidity of surroundings, ultraviolet radiation, and some kinds of chemic contamination.By the way of RT-PCR and cell culture of air samples from two hospitals we can offer effective advise about controlling SARS-CoV airborne infection. We used Andersen microbial sampler 2 stage (AMS-2) to sample incessantly at five spots (Corridors, Exhaust fans, cleaning-districts, sickrooms and sickroom-balcony) and obtained 112 air samples in different times. Then we accomplished washing of air samples, isolation of total RNA, RT-PCR, Vero-E6 cell culture and gene-sequence analysis. We have successfully separated active SARS-CoV (called LK309 strain) from air samples and SARS-CoV RNA was detected at all five spots in two hospitals, among which the detecting positive rate is 25 (52. 1%) of 48 air samples from sickrooms in XiaoTang-hill hospital and is 9 (29. 03%) of 31 ones from sickrooms in PLA 309 hospital, the results showed that convalescence patients still exhale viruses by respiratory tract and at 5 to 10m distance
本研究旨在通过对SARS病人定点收治医院(小汤山医院、309医院)室内空气SARS-CoV污染的研究,为更好地控制SARS-CoV的空气传播感染提供实验依据。实验采用FA-Ⅱ型空气微生物采样器在医院病房区、病房阳台、内走廊、排风扇和护士站五个地点持续采样,之后进行空气样本的洗脱、Veto-E6细胞培养、RT-PCR及序列测定。其结果病房区、病房阳台、内走廊、排风扇和清洁区五个地点均有SARS-CoV RNA的检出,其中以小汤山医院排气口下风向的阳性率最高(58.3%),护士站相对较低(25.0%);从309医院一名确诊为SARS病人病房内的空气样本中分离出一株活性SARS-CoV(称之为LK309株),并稳定传代;与恢复期SARS病人血清免疫荧光染色阳性,RT-PCR扩增产物的测序结果显示LK309株与已知SARS-CoY的同源性在98%以上。相关数据表明SARS病人定点收治医院空气中SARS-CoV的污染比较严重,急性后期的病人仍然从呼吸道大量排毒;但在病房室内外的空气中几乎没有检测到活的SARS-CoV,初步评估认为,SARS病人病房采取通风和消毒的措施对降低室内污染程度是非常有益的;而空气消毒和环境因素对SARS病毒的存活有较大的影响,故SARS病人病房排出的空气对周边环境造成的危害很小。
针对SARS-CoY空气分离株LK309,根据引物步移法的原则,通过覆盖全基因
SARS-CoV is a kind of novel single RNA virus which can arose zoonosis. This kind of virus can spread quickly via short-haul airborne infection and hand-in-hand infection. In hospital and at home SARS-CoV takes on prominent assembly phenomena. Admittedly airborne infection can be interfered by multi-fold factors, the most important factor is livability of SARS-CoV in the aerosol state, and the others include temperature and humidity of surroundings, ultraviolet radiation, and some kinds of chemic contamination.By the way of RT-PCR and cell culture of air samples from two hospitals we can offer effective advise about controlling SARS-CoV airborne infection. We used Andersen microbial sampler 2 stage (AMS-2) to sample incessantly at five spots (Corridors, Exhaust fans, cleaning-districts, sickrooms and sickroom-balcony) and obtained 112 air samples in different times. Then we accomplished washing of air samples, isolation of total RNA, RT-PCR, Vero-E6 cell culture and gene-sequence analysis. We have successfully separated active SARS-CoV (called LK309 strain) from air samples and SARS-CoV RNA was detected at all five spots in two hospitals, among which the detecting positive rate is 25 (52. 1%) of 48 air samples from sickrooms in XiaoTang-hill hospital and is 9 (29. 03%) of 31 ones from sickrooms in PLA 309 hospital, the results showed that convalescence patients still exhale viruses by respiratory tract and at 5 to 10m distance
引文
1、World Health Organization. Acute respiratory syndrome in China-Upd ate 2:disease outbreak reported, http://www.who.int/csr/don/2003-2-14/en/May2003.
2、http://www.who.int/csr/sars/country/table2003_09_23/en/.
3、It can be available at http://www.who.int/csr/sars/.
4、Hsu L Y, Lee C , Green J A, Ang B, Paton N I, Lee L, Villacian J S, Lim P L, Earnest A, Leo Y S. Severe acute respiratory syndrome (SARS) in Singapore: clinical features of index patient and initial contacts. Emerg Infect Dis, 2003 Jun, 9(6):713-7.
5、It can be available at http://www.who.int/csr/sars/archive/2003_03_15/en/.
6、段宁,刘景洋,乔琦.北京市SARS病例数与空气污染指数的相关性探讨.环境科学研究,2003,Vol.16,No.3:40-43.
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14, American Conference of Governmental Industrial Hygienists (ACGIH) : Bioaerosols: Assessment and Control. Edited by Macher J, 1999.
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18, Higgins J A, Cooper M, Schroeder Tucker L, Black S, Miller J D, Karns J S, Manthey E, Breeze R, Perdue M L. A field investigation of Bacillus anthracis contamination of US Department of Agriculture and other Washington DC buildings during the anthrax attack of October 2001. Appl Environ Microbiol, 2003, 69:593-599.
19, Buttner M P, Stetzenbach L D. Monitoring of fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl Environ Microbiol, 1993, 59:219-226. 19
20, Weis C P, Intrepido A J, Miller A K, Cowin P G, Durno M A, Gebhardt J S, Bull R. Secondary aerosolization of viable Bacillus anthracis spores in contaminated US Senate office. J Am Med Assoc, 288:2853-2858.
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2、http://www.who.int/csr/sars/country/table2003_09_23/en/.
3、It can be available at http://www.who.int/csr/sars/.
4、Hsu L Y, Lee C , Green J A, Ang B, Paton N I, Lee L, Villacian J S, Lim P L, Earnest A, Leo Y S. Severe acute respiratory syndrome (SARS) in Singapore: clinical features of index patient and initial contacts. Emerg Infect Dis, 2003 Jun, 9(6):713-7.
5、It can be available at http://www.who.int/csr/sars/archive/2003_03_15/en/.
6、段宁,刘景洋,乔琦.北京市SARS病例数与空气污染指数的相关性探讨.环境科学研究,2003,Vol.16,No.3:40-43.
7、Mukoda T J, Todd L A, Sobsey M D. PCR and gene probe for detecting bioaerosols. J Aerosol Sci, 25:1523.
8、Alvarez A J, Buttner M P, Toranos G A, et al. Use of solid-phase PCR for enhanced detection of airborne microbioorganisms. Appl Enviro Microbiol, 1994, 60:374-376.
9、Alvarez A J, Buttner M P, Stetzenbach L D. PCR for bioaerosol monitoring: sensitivity and environmental interference. Appl Enviro Microbiol, 1995, 61:3639-3644.
10、Sawyer M H, Chamberlin C J, Wu Y N, et al. Detection of varicella-zoster virus DNA in air samples from hospital rooms. J Infect Dis, 1994, 169:91-94.
11、McCluskey R, Sandin R, Greene J. Detection of airborne cytomegalovirus in hospital room of immuncompromised patients. J Virol Methods, 1996,56:115-118.
12, Cox C S, Wathes C M. Bioaerosols in the environment. In Bioaerosols Handbook. Boca Raton, FL: Lewis Publishers; Edited by CS Cox and CM Wathes, 1995:11-14.
13, Lighthart B. Physics of bioaerosols. In Atmospheric Microbial Aerosols: Theory and Applications. Edited by Lighthart B, Mohr J. New York: Chapman and Hall, 1994:5-27.
14, American Conference of Governmental Industrial Hygienists (ACGIH) : Bioaerosols: Assessment and Control. Edited by Macher J, 1999.
15, Radosevich J L, Wilson W J, Shin J H, DeSantis T Z, Andersen G L. Development of a high-volume aerosol collection system for the identification of air-borne micro-organisms. Lett Appl Microbiol,2002, 34:162-167.
16, Buttner M P, Stetzenbach L D. Evaluation of four aerobiological sampling methods for the retrieval of aerosolized Pseudomonas syringae. Appl Environ Microbiol, 1991, 57:1268-1270.
17, Crook B. Inertial samplers: biological perspectives. In Bioaerosols Handbook. Edited by Cox CS, Wathes CM. Boca Raton, FL: Lewis Publishers; 995,247-267.
18, Higgins J A, Cooper M, Schroeder Tucker L, Black S, Miller J D, Karns J S, Manthey E, Breeze R, Perdue M L. A field investigation of Bacillus anthracis contamination of US Department of Agriculture and other Washington DC buildings during the anthrax attack of October 2001. Appl Environ Microbiol, 2003, 69:593-599.
19, Buttner M P, Stetzenbach L D. Monitoring of fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl Environ Microbiol, 1993, 59:219-226. 19
20, Weis C P, Intrepido A J, Miller A K, Cowin P G, Durno M A, Gebhardt J S, Bull R. Secondary aerosolization of viable Bacillus anthracis spores in contaminated US Senate office. J Am Med Assoc, 288:2853-2858.
21, Saiki R K, Scharf S, Faloona F, Mullis K B, Horn G T, Erlich H A, Arnheim N. Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 1985, 230:1350-1354. 21
22, Alvarez A J, Buttner M P, Toranzos G A, Dvorsky E A, Toro A, Heikes T B, Mertikas L E, Stetzenbach L D. The use of solid-phase polymerase chain reaction for the enhanced detection of airborne microorganisms. Appl Environ Microbiol, 1994, 60:374-376. 21
23, Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med, 1998, 36:255-269.
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