公共场所水环境中活嗜肺军团菌的快速检测方法
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摘要
旨在建立一种基于16S rRNA前体的分子学检测方法用于快速检测活嗜肺军团菌,应用该方法与ISO标准法调查公共场所水环境中嗜肺军团菌的水平及污染现状。此研究方法的理论基础在于营养刺激后可诱导嗜肺军团菌16S rRNA前体的合成,后者可作为检测细胞活性的指标。分别应用预刺激RT-qPCR法和ISO法对嗜肺军团菌、非嗜肺军团菌以及非军团菌进行检测,并验证两种方法的特异性、灵敏度。应用预刺激RT-qPCR法和ISO法检测公共场所水环境中的嗜肺军团菌,比较两者结果的一致性。结果显示,预刺激时间大于3 h时,嗜肺军团菌16S rRNA前体的增长缓慢,最佳预刺激时间设置为3 h。预刺激RT-qPCR法与ISO法均能较好地检出嗜肺军团菌,特异性均为100%,预刺激法的最低检测浓度(Limit of detection,LOD)为102Cell/L,ISO标准法的LOD为104 Cell/L。预刺激法及ISO法的总阳性率分别为43.5%(30/69)和40.6%(28/69),两种方法的检出率差异无明显统计学意义(C2=0.119,P=0.730)。预刺激RT-qPCR法是一种快速有效的检测活嗜肺军团菌的方法,诊断灵敏度及特异性高,是ISO标准法潜在的替代方案。
This work aims to establish a molecular detection method for the rapid detection of viable Legionella pneumophila based on16 S rRNA precursor,and the contamination level and status of L. pneumophila in the water environment of public places may be detected by this method and ISO method. The established method is based on the synthesis of 16 S rRNA precursor in L. pneumophila via a nutritional stimulation,which is an indicator for cells' viability. Pre-stimulated RT-qPCR method and ISO method were used to detect L. pneumophila and non-Legionella pneumophila,as well as other non-Legionella,and the specificity and sensitivity of these two methods were validated.Further,the pre-stimulated RT-q PCR method and ISO method were employed to detect L. pneumophila in the water environment of public places,and the consistency of the results between the 2 methods were compared.The 16 S rRNA precursor of L. pneumophila increased slowly when the prestimulation time was > 3 h,thus the optimal pre-stimulation time was set to be 3 h. L. pneumophila were well detected by both pre-stimulated RT-qPCR method and ISO method,and the specificity for both was 100%. The Limit of Detection(LOD)by pre-stimulated RT-qPCR method was 102 cells/L,while LOD by ISO method was 104 cells/L. The positive rate of L. pneumophila in the water environment of public places was43.5%(30/69)by pre-stimulated RT-qPCR method and 40.6%(28/69)by ISO method,and the difference of detection by both methods was of no statistical significance(C2=0.119,P=0.730). In conclusion,pre-stimulated RT-qPCR method is a rapid and effective method for detecting L. pneumophila with high sensitivity and specificity,and it can be used as a potential alternative to the ISO method.
This work aims to establish a molecular detection method for the rapid detection of viable Legionella pneumophila based on16 S rRNA precursor,and the contamination level and status of L. pneumophila in the water environment of public places may be detected by this method and ISO method. The established method is based on the synthesis of 16 S rRNA precursor in L. pneumophila via a nutritional stimulation,which is an indicator for cells' viability. Pre-stimulated RT-qPCR method and ISO method were used to detect L. pneumophila and non-Legionella pneumophila,as well as other non-Legionella,and the specificity and sensitivity of these two methods were validated.Further,the pre-stimulated RT-q PCR method and ISO method were employed to detect L. pneumophila in the water environment of public places,and the consistency of the results between the 2 methods were compared.The 16 S rRNA precursor of L. pneumophila increased slowly when the prestimulation time was > 3 h,thus the optimal pre-stimulation time was set to be 3 h. L. pneumophila were well detected by both pre-stimulated RT-qPCR method and ISO method,and the specificity for both was 100%. The Limit of Detection(LOD)by pre-stimulated RT-qPCR method was 102 cells/L,while LOD by ISO method was 104 cells/L. The positive rate of L. pneumophila in the water environment of public places was43.5%(30/69)by pre-stimulated RT-qPCR method and 40.6%(28/69)by ISO method,and the difference of detection by both methods was of no statistical significance(C2=0.119,P=0.730). In conclusion,pre-stimulated RT-qPCR method is a rapid and effective method for detecting L. pneumophila with high sensitivity and specificity,and it can be used as a potential alternative to the ISO method.
引文
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[18]MauchlineWS,JamesBW,FitzgeorgeRB,etal.Growth temperature reversibly modulates the virulence of Legionella pneumophila[J]. Infection and Immunity, 1994, 62(7):2995-2997.
[19]Mampel J, Spirig T, Weber SS, et al. Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions[J].Appl environ Microbiol, 2006, 72(4):2885-2895.
[20]莫自耀,秦建强,赵红波,等.应用单一和双重荧光定量PCR法快速检测军团菌[J].中华生物医学工程杂志, 2011, 1:61-65.
[21]Boss R, Baumgartner A, Kroos S, et al. Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT-PCRbased method[J]. J Appl Microbiol, 2018, 125(4):1216-1225.
[22]Beaute J, Zucs P, De Jong B, et al. Legionnaires disease in Europe,2009-2010[J]. Euro Surveillance, 2013, 18(10):20417.
[23]Omiccioli E, Schiavano GF, Ceppetelli V, et al. Validation according to ISO/TS 12869:2012 of a molecular method for the isolation and quantification of Legionella spp. in water[J].Molecular and Cellular Probes, 2015, 29(2):86-91.
[24]Bonetta S, Bonetta S, Ferretti E, et al. Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods[J]. J Appl Microbiol, 2010, 108(5):1576-1583.
[25]Greenhalgh T. How to read a paper. Papers that report diagnostic or screening tests[J]. BMJ, 1997, 315(7107):540-543.
[2]Parr A, Whitney EA, Berkelman RL. Legionellosis on the rise:A review of guidelines for prevention in the united states[J]. J Public Health Manag Pract, 2015, 21(5):E17-26.
[3]Rhoads WJ, Ji P, Pruden A, et al. Water heater temperature set point and water use patterns influence Legionella pneumophila and associated microorganisms at the tap[J]. Microbiome, 2015, 3:67.
[4]Lanternier F, Ader F, Pilmis B, et al. Legionnaire’s disease in compromised hosts[J]. Infectious Disease Clinics of North America, 2017, 31(1):123-135.
[5]Bedard E, Fey S, Charron D, et al. Temperature diagnostic to identify high risk areas and optimize Legionella pneumophila surveillance in hot water distribution systems[J]. Water Research, 2015, 71:244-256.
[6]Kirschner AKT. Determination of viable legionellae in engineered water systems:Do we find what we are looking for?[J]. Water Research, 2016, 93:276-288.
[7]Al-Bana BH, Haddad MT, Garduno RA. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells[J]. Environmental Microbiology, 2014,16(2):382-395.
[8]Li L, Mendis N, Trigui H, et al. Transcriptomic changes of Legionella pneumophila in water[J]. BMC Genomics, 2015, 16:637.
[9]Ducret A, Chabalier M, Dukan S. Characterization and resuscitation of ‘non-culturable’ cells of Legionella pneumophila[J]. BMC Microbiology, 2014, 14:3.
[10]Yanez MA, Nocker A, Soria-Soria E, et al. Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR[J]. Journal of microbiological methods,2011, 85(2):124-30.
[11]Cangelosi GA, Weigel KM, Lefthand-Begay C, et al. Molecular detection of viable bacterial pathogens in water by ratiometric prer RNA analysis[J]. Appl environ Microbiol, 2010, 76(3):960-2.
[12]Cangelosi GA, Brabant WH. Depletion of pre-16S rRNA in starved Escherichia coli cells[J]. J Bacteriol, 1997, 179(14):4457-4463.
[13]Stroot PG, Oerther DB. Elevated precursor 16S r RNA levels suggest the presence of growth inhibitors in wastewater[J]. Water Science and Technology, 2003, 47(11):241-250.
[14]Lu T, Stroot PG, Oerther DB. Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment[J]. Appl environ Microbiol, 2009, 75(13):4589-4598.
[15]Warren JD, Xiong W, Bunker AM, et al. Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer[J].BMC Medicine, 2011, 9:133.
[16]张宝莹,刘凡,陈逊,等.应用两种PCR法快速检测公共场所水环境中嗜肺军团菌[J].中国卫生检验杂志, 2014, 24(14):1984-1986, 1990.
[17]Warren WJ, Miller RD. Growth of Legionnaires disease bacterium(Legionella pneumophila)in chemically defined medium[J]. J Clin Microbiol, 1979, 10(1):50-55.
[18]MauchlineWS,JamesBW,FitzgeorgeRB,etal.Growth temperature reversibly modulates the virulence of Legionella pneumophila[J]. Infection and Immunity, 1994, 62(7):2995-2997.
[19]Mampel J, Spirig T, Weber SS, et al. Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions[J].Appl environ Microbiol, 2006, 72(4):2885-2895.
[20]莫自耀,秦建强,赵红波,等.应用单一和双重荧光定量PCR法快速检测军团菌[J].中华生物医学工程杂志, 2011, 1:61-65.
[21]Boss R, Baumgartner A, Kroos S, et al. Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT-PCRbased method[J]. J Appl Microbiol, 2018, 125(4):1216-1225.
[22]Beaute J, Zucs P, De Jong B, et al. Legionnaires disease in Europe,2009-2010[J]. Euro Surveillance, 2013, 18(10):20417.
[23]Omiccioli E, Schiavano GF, Ceppetelli V, et al. Validation according to ISO/TS 12869:2012 of a molecular method for the isolation and quantification of Legionella spp. in water[J].Molecular and Cellular Probes, 2015, 29(2):86-91.
[24]Bonetta S, Bonetta S, Ferretti E, et al. Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods[J]. J Appl Microbiol, 2010, 108(5):1576-1583.
[25]Greenhalgh T. How to read a paper. Papers that report diagnostic or screening tests[J]. BMJ, 1997, 315(7107):540-543.