环境水中嗜肺军团菌的分离培养法和巢式PCR法比较

目的应用传统分离培养法和巢式PCR法检测环境水中嗜肺军团菌污染状况,并比较两种方法的检测效果。方法于2012年7-10月,采集北京市丰台区商场和超市、宾馆和饭店、综合性医院三类场所的中央空调冷却水、自来水、淋浴水、景观水,分别使用培养法及巢式PCR法检测嗜肺军团菌。结果共检测187件水样,传统分离培养法对嗜肺军团菌的检出率为2.67%(5/187),低于巢式PCR法[19.79%(37/187)],差异有统计学意义(χ~2=27.465,P0.01)。结论北京丰台区多种环境水体存在嗜肺军团菌污染;巢式PCR法对环境水中嗜肺军团菌的灵敏度优于传统分离培养法。     

常州市22家宾馆集中空调水系统及淋浴水嗜肺军团菌污染状况分析

目的研究常州市宾馆集中空调水系统及淋浴水中嗜肺军团菌的污染状况。方法 2015年5月至10月对22家宾馆的集中空调水系统及淋浴水进行采样,培养法和巢式PCR方法检测水样中嗜肺军团菌的污染状况。结果经过培养法检测,冷却水、冷凝水、淋浴水中嗜肺军团菌的阳性检出率分别为39.5%(17/43)、30%(6/20)、23.1%(6/26),检出率差异无统计学意义(χ~2=2.076,P=0.354),分离菌株中LP1型为主,占比60.7%,培养法和巢式PCR方法水样中嗜肺军团菌检出率分别为33.3%(23/69)、43.5%(30/69),差异有统计学意义(χ~2=9.554,P=0.002)。结论检测的空调水系统及淋浴水均存在不同程度的嗜肺军团菌污染,需加强对宾馆水环境进行嗜肺军团菌污染的监测,预防军团病的发生。     

广东新会地区公共环境冷却塔水系军团菌污染状况调查

目的查明广东新会地区公共环境冷却塔水系军团菌分布状况,为预防和控制军团菌病暴发流行提供依据。方法自2004-2006年8～10月,在新会区各大宾馆、饭店、学校、医院和公共娱乐场所共采集中央空调冷却塔水样112份,进行军团菌分离培养和PCR快速鉴定。结果在新会区采集的112份公共环境中央空调冷却塔水样中分离培养检出嗜肺军团菌26株,检出率23.2%;PCR直接检测嗜肺军团菌(LP-DNA)阳性57份,阳性率50.9%。结论在广东新会地区环境水源中嗜肺军团菌污染具有普遍性,PCR检测嗜肺军团菌是一种快速、敏感、特异性良好的检测法。     

嗜肺军团菌荧光定量PCR法的建立和初步应用

目的建立嗜肺军团菌特异、快速检测方法,探讨南京地区公共场所环境水样嗜肺军团菌污染状况。方法根据嗜肺军团菌的mip基因保守序列设计引物和探针,通过对嗜肺军团菌标准株ATCC33152、嗜肺军团菌南京分离株及大肠埃希菌ATCC25922、鼠伤寒沙门菌ATCC14028、福氏2a志贺菌ATCC12022进行检测,验证该方法的特异性。并应用该法对南京地区某商场、宾馆、办公楼、医院共计13家的中央空调系统的冷却水、淋浴水和景观水共计88份环境水样进行了PCR检测及军团菌的分离培养。结果该法对嗜肺军团菌有特异性扩增曲线,而对其他细菌无阳性扩增曲线。88份水样中荧光定量PCR方法检测出28份阳性,阳性率为31.8%;传统培养方法分离出17株军团菌,分离率为19.3%。结论本文建立的荧光定量PCR检测方法具有良好的特异性,可用于环境水中军团菌污染状况调查。南京地区中央空调冷却水中检出率最高,存在发生军团菌病的可能,应引起足够重视。     

空气中嗜肺军团菌的巢式PCR和荧光定量PCR测定方法比较

目的评价巢式PCR和荧光定量PCR方法在嗜肺军团菌气溶胶检测中的应用。方法于2012年8—10月在北京、南京、苏州和深圳四城市选取38家公共场所,使用带有虚拟浓缩器的Bio-sampler生物冲击式采样器采集气溶胶样品,分别应用巢式PCR和荧光定量PCR法检测样品中的嗜肺军团菌。结果共采集气溶胶样品165份。巢式PCR法测定嗜肺军团菌的阳性率为8.5%(14/165),荧光定量PCR法测定嗜肺军团菌的阳性率为26.1%(43/165),后者高于前者,差异有统计学意义(P0.05)。结论采用荧光定量PCR法测定空气中嗜肺军团菌的灵敏度优于巢式PCR法。     

北京市西城区淋浴热水中嗜肺军团菌污染状况调查

目的了解北京市西城区淋浴热水系统军团菌污染状况。方法随机选取淋浴热水水样130份,进行军团菌的分离培养与鉴定,再以聚合酶链反应(PCR)方法进行复核。结果 130份水样检出嗜肺军团菌阳性28件,总体阳性率为21.5%。7—11月嗜肺军团菌的阳性率差异有统计学意义。血清分型结果总体以LP1型和LP7型为主。结论西城区热水环境中嗜肺军团菌污染严重,应加强监测管理,并尽快采取预防控制措施以保护广大人群身体健康。     

泰州市116份公共场所集中空调冷却水嗜肺军团菌监测结果

目的 为了解泰州市公共场所集中空调冷却水中嗜肺军团菌污染状况,为军团病的防控提供数据。方法 于2013-2015年采集了泰州市公共场所集中空调冷却水116份,进行了嗜肺军团菌分离培养,同时进行嗜肺军团菌核酸检测。结果 116份冷却水样本中26份分离培养出嗜肺军团菌,检出率为22.4%,荧光PCR法嗜肺军团菌核酸检测结果31份核酸阳性,检出率为26.7%。宾馆(饭店)、商场(超市)冷却水样中分离培养检出率分别为19.7%、27.5%,荧光PCR法嗜肺军团菌核酸阳性检出率分别为22.3%、35.0%。结论 泰州公共场所的集中空调冷却水嗜肺军团菌污染情况严重,已对长期在此环境下工作人群健康构成潜在威胁。     

传统细菌培养法与荧光PCR法分离公共场所嗜肺军团菌结果比较

目的：对公共场所环境样本（水、土壤、气溶胶）进行嗜肺军团菌监测,掌握南京市公共场所中军团菌的污染情况和菌型分布。方法：对12家商场、医院中央空调系统采集水样（冷却水、景观水、淋浴水、自来水）,土壤类样品（背景土、花卉土、风管积尘、景观土）,采用传统细菌培养法及荧光PCR法进行嗜肺军团菌的分离鉴定。结果：12家单位共采样234份（水样88份、土壤146份）,传统细菌培养法检出嗜肺军团菌9份（主要类型为Lp1型）,检出率为3.85%;而实时荧光PCR法检出47份阳性（主要类型为Lp1型）,检出率为20.08%。两者检出阳性率比较差异有统计学意义（P〈0.05）。结论：南京市公共场所存在嗜肺军团菌污染,荧光PCR法总检出率明显高于传统细菌培养法,荧光PCR方法较培养法灵敏性较好。     

空调风管内壁积尘中的嗜肺军团菌巢式PCR检测研究

目的应用巢式PCR方法检测集中空调风管内壁积尘中嗜肺军团菌的污染情况。方法于2009年8月随机抽取上海市4个城区集中空调冷却水嗜肺军团菌培养法阳性的公共场所,采集集中空调风管内壁积尘。巢式PCR方法扩增嗜肺军团菌种Mip基因,琼脂糖凝胶电泳检测扩增产物。结果共采集空调风管内壁积尘40份,嗜肺军团菌阳性样品11份,阳性率为27.5%(11/40)。4个城区风管内壁积尘中嗜肺军团菌阳性率的差异没有统计学意义(χ2=7.446,P=0.064)。当冷却塔与新风口直线距离处于0～4m范围内,风管内壁积尘中嗜肺军团菌的阳性率随距离增加有升高趋势。结论巢式PCR方法检测积尘中嗜肺军团菌特异性较好,集中空调风管内壁积尘中存在嗜肺军团菌污染。     

公共场所集中空调水系统中嗜肺军团菌传统分离和PCR检测

目的了解江干区公共场所集中空调水系统中嗜肺军团菌的污染状况,比较传统分离和PCR 2种检测方法的检出率。方法依据WS 394—2012《公共场所集中空调通风系统卫生规范》对样品进行分离培养及鉴定;同时对样品进行荧光定量PCR检测。结果共采集20份样品,传统分离和PCR法洗脱液中嗜肺军团菌检出率分别为10.0%和45.0%,2种方法检出率差异有统计学意义(P0.05);冷却水中嗜肺军团菌检出率显著高于冷凝水;分离出38株嗜肺军团菌,其血清型以LP7和LP1为主,分别占44.7%和21.1%。结论本区集中空调水系统嗜肺军团菌污染较严重,应加强冷却水塔的规范清洗消毒。在嗜肺军团菌检测中,传统分离和PCR检测可根据需要结合使用。     

呼吸道感染患者军团菌抗体水平调查

目的了解呼吸道感染患者嗜肺军团菌（Lp1-Lp6型）及米克戴德军团菌（Lm型）抗体水平,以掌握深圳地区军团菌感染状况。方法收集医院呼吸道感染患者血清标本56份,采用微量凝集试验的方法,检测嗜肺军团菌（Lp1-Lp6型）及米克戴德军团菌（Lm型）的抗体效价。结果 56份呼吸道感染患者血清未检出嗜肺军团菌（Lp1-Lp6型）抗体阳性;检出米克戴德军团菌Lm型抗体阳性6份,阳性率10.7%。结论深圳地区存在米克戴德军团菌（Lm型）感染,对不明原因呼吸道感染患者应重视军团菌病原菌及抗体的检测。     

Soil as a source of Legionella pneumophila serogroup 1 (Lp1)

OBJECTIVE: To investigate the potential source of a case of Legionnaires' disease caused by an unusual serotype of Legionella pneumophila serogroup 1 (Lp1) in regional Victoria in May 2001. METHOD: Epidemiological and environmental investigation of the source of infection of a case of Legionnaires' disease in regional Victoria in May 2001. RESULTS: Extensive environmental investigations did not reveal any cooling water tower systems close to the residence or the shopping centre that the case visited prior to illness. The sputum culture and a soil sample from the field at the plant nursery where the case worked prior to illness were both positive for Legionella pneumophilia serogroup 1, MDU pulsovar 97:103. CONCLUSION: Legionella pneumophila has been found in soil and was further found to be associated with a case of Legionella pneumophila. IMPLICATIONS: Public health authorities should consider exposures to soil and potting mixes when investigating cases of Legionella pneumophila where the case has no apparent association with cooling towers. Safe gardening practices should be promoted among the community.     

深圳市公共场所中军团菌污染状况

目的调查深圳市公共场所中军团菌的污染状况。方法采集公共场所中水、土壤、风管积尘和气溶胶样本,按照＂卫生行业专项任务工作方案＂检测军团菌和嗜肺军团菌。结果军团菌阳性率水样为43.3%、气溶胶为3.85%、积尘为0.90%、土壤为0;嗜肺军团菌阳性率水样为36.8%、气溶胶为0%、积尘为0.90%、土壤为0。深圳市公共场所中分离的76株嗜肺军团菌有7种血清型,优势血清型为LP1和LP3,分别占57.9%和25.0%。部分水样存在着多种菌型共生的现象。结论深圳公共场所水环境中嗜肺军团菌污染严重,综合性医院空气中军团菌阳性率较高,公共场所存在军团菌病暴发的隐患。     

Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.     

Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.     

嗜肺军团菌荧光定量PCR检测方法的建立

目的：建立嗜肺军团菌mip基因荧光定量PCR检测方法。方法：利用Primer Express软件设计特异性引物和探针,对质粒标准品、嗜肺军团菌、非嗜肺军团菌及其他菌株进行检测,验证方法的特异性、敏感性和重复性,最后采集环境样本进行检测。结果：该方法特异性好,嗜肺军团菌呈现阳性结果,而非嗜肺军团菌及其他菌株均为阴性结果。检测灵敏度达293copies／μl;重复性较好,Ct值变异系数较小;检测的12份环境样本中5份阳性。结论：该方法快速且具有较好的特异性、敏感性、重复性,适于外环境嗜肺军团菌污染调查及应急事件的快速检测。     

Legiolert酶底物法与传统培养法对公共场所水体样本中嗜肺军团菌检出结果的比较

目的比较Legiolert酶底物法与传统培养法对公共场所水体样本中嗜肺军团菌的检出效果。方法分别利用Legiolert酶底物法与传统培养法对采集于公共场所的冷却水、淋浴水样本进行嗜肺军团菌检测,并对分离株进行验证及血清分型。结果共采集并检测样品68份,传统培养法与Legiolert酶底物法的检出率分别为5.88%(4/68)及35.29%(24/68),差异有统计学意义(χ2=16.41,P<0.01)。两种方法的检测符合率为64.71%(44/68),检测效果的差异有统计学意义(配对χ2=16.41,P=0.000)。分离出的25株嗜肺军团菌血清型以LP1为主(14/25)。结论Legiolert酶底物法对公共场所水体样本中嗜肺军团菌的检出率高于传统培养法。     

深圳市6家医院集中空调冷却水军团菌污染状况及其管理对策

目的了解深圳市福田区医院集中空调冷却水军团菌污染现况,为医院集中空调管理提供依据。方法根据《公共场所集中空调通风系统卫生规范》,采集辖区内6家医院16个大型空调冷却塔共32份冷却塔水进行军团菌检测。结果所调查的医院空调冷却塔水中军团菌检出率为50.0%,共分离出16株军团菌,均为LPl型嗜肺军团菌。结论军团菌广泛存在于医院空调冷却塔水中,对医院内的易感人群的健康造成潜在威胁,对这一潜在传染来源应采取有效防范措施。