套式聚合酶链反应法检测军团菌

应用套式聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）技术，建立了扩增嗜肺军团菌巨噬细胞感染增强因子（ｍｉｐ）基因序列的方法。对１４株标准军团菌和５株标准大肠埃希氏菌等进行检测。结果仅嗜肺军团菌扩增出６４９ｂｐ特异性片段，扩增灵敏度为１００ＣＦＵ／ｍｌ。对１７份人血标本进行检测，表明军团菌ｎｅｓｔ－ｅｄ－ＰＣＲ的敏感性明显高于血清学检测和细菌培养     

军团菌16SrRNA聚合酶链反应方法的建立及应用

建立了１６ＳｒＲＮＡ基因检测军团菌的多聚酶链反应方法。扩增参考菌株染色体ＤＮＡ（Ｌ．Ｐｎｅｕｍｏｐｈｉｌａ１～１４、Ｌ．ｂｏｚｅｍａｎｎｉ、Ｌ．ｄｕｍｏｆｉ、Ｌ．ｌｏｎｇｂｅａｃｈａｅ、Ｌ．ｍｉｃｄａｄｅｉ、Ｌ．ｊｏｒｄａｎｉｓ）,均可检出出３８６ｂｐ的基因片段,敏感性为１０３ｃｆｕ／ｍｌ（平均值）;而扩增９株非军团菌均为阴性。对模拟血标本的检测,其敏感性可达１０２ｃｆｕ／ｍｌ。应用本检测系统检测了２４例临床标本,包括１９份血、５份胸水,阳性率为７０．８％（１７／２４）。与血清学检测和临床诊断治疗结果相符合。上述结果表明该方法敏感、特异、快速、简便。     

巢式聚合酶链反应检测伤寒沙门氏菌

本文采用扩增伤寒沙门氏菌鞭毛抗原基因Ⅵ区一段特异性片段的两对引物，用ＰＣＲ法检测伤寒沙门氏菌与非伤寒沙门氏菌，结果表明阳性符合率达１００％，且反应体系中达１０２ｃｆｕ／ｍｌ浓度时，伤寒沙门氏菌即可检出。检查伤寒确诊患者粪便标本４份，ＰＣＲ扩增均阳性。     

小儿军团菌病的快速检验及临床特征

目的:探索快速准确的诊断军团菌感染的方法,了解儿童军团菌感染的临床特点。方法:根据嗜肺军团菌巨噬细胞感染增强子(m ip)基因建立一种检测嗜肺军团菌(LP)PCR方法,用该方法对65例呼吸系统炎症患儿的痰及咽拭子进行PCR检测,用传统血清学TAT方法检测嗜肺军团菌各型抗体。结果:嗜肺军团菌1~14型均扩增出628 bp的特异性片段,而非嗜肺军团菌LM及肺炎球菌均未见扩增片段;敏感性为2.1 pg/μl的嗜肺军团菌DNA。65例临床标本中6例扩增出628 bp的基因片段,阳性率为9.23%,其中4例应用TAT方法检测Lp抗体也为阳性。此6例患儿临床均为肺炎。结论:①利用嗜肺军团菌巨噬细胞感染增强子(m ip)基因建立的检测嗜肺军团菌PCR方法特异性高,敏感性强,适用标本范围广,在嗜肺军团菌早期诊断中有一定价值。②在儿科呼吸系统感染性疾病中,存在嗜肺军团菌感染,在本资料中,65例呼吸道感染患儿,PCR方法检出6例,嗜肺军团菌感染阳性率为9.23%,而TAT方法检出4例,阳性率为6.15%。说明PCR方法敏感性高于TAT方法。③嗜肺军团菌感染在临床常表现为以重症肺炎、难治性肺炎为主的全身性感染,临床表现不典型,易误诊、漏诊,应引起儿科医师的警惕与重视。     

聚合酶链反应标记输血传播病毒(TTV)地高辛素探针的初步应用

用聚合酶链反应法（ＰＣＲ）制备地高辛素标记的输血传播病毒（ＴＴＶ）探针，并与巢式ＰＣＲ法（ｎＰＣＲ）比较。结果该探针具有ＴＴＶ特异性，其灵敏度为１０ｐｇＤＮＡ。应用该法检测１０８份非甲～庚型肝炎病人的血清标本，ＴＴＶＤＮＡ阳性率为１８５％（２０／１０８）；检测２２份病人的粪便标本，ＴＴＶＤＮＡ阳性率为２７３％（６／２２）。该法与ｎＰＣＲ法的总符合率为９６９％（１２６／１３０）。结论：用ＰＣＲ法直接制备地高辛素标记ＤＮＡ探针简便、快速、灵敏度高，特异性好。应用该法从６名病人的粪便中检出ＴＴＶＤＮＡ，提示ＴＴＶ有可能通过粪口途径传播。     

动脉粥样硬化血管组织中的巨细胞病毒感染

为探讨巨细胞病毒（Ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ，ＣＭＶ）感染与动脉粥样硬化的相关性，应用聚合酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ，ＰＣＲ）技术及二次ＰＣＲ法对病例组２６例动脉粥样硬化血管组织石蜡标本和对照组２３例正常血管组织石蜡标本进行检测。结果：应用传统ＰＣＲ技术检测到病例组１７例ＣＭＶＤＮＡ阳性，阳性率为６５４％（１７／２６），对照组５例阳性，阳性率为２１７％（５／２３）；应用二次ＰＣＲ法检测到病例组２０例阳性，阳性率７６９％（２０／２６），对照组８例阳性，阳性率３４８％（８／２３）。结果表明，ＣＭＶ潜伏感染与动脉粥样硬化具有密切联系     

聚合酶链反应标记输血传播病毒（TTV）地高辛霉探针的初步应用

用聚合酶链反应法（ＰＣＲ）制备地高辛素标记的输血传播病毒（ＴＴＶ）探针,并与巢式ＰＣＲ法比较。结果 该探针具有ＴＴＶ特异性,其灵敏度为１０ｐｇ ＤＮＡ。应用该法检测１０８份非甲￣庚型肝炎病人的血清标本,应用该法检测１０８份非甲￣庚型肝炎病人的血清标本,ＴＴＶ ＤＮＡ阳性率为１８．５％（２０／１０８）;检测２２份病人的粪便标本,ＴＴＶ ＤＮＡ阳性率为２７．３％（６／２２）。该法与ｎＰＣＲ法的总符合率     

基于ＴａｑＭａｎ探针三重荧光ＰＣＲ 检测沙门菌、肠炎沙门菌和鼠伤寒沙门菌

摘要：目的　建立基于ＴａｑＭａｎ探针三重荧光ＰＣＲ 检测沙门菌（狊犪犾犿狅狀犲犾犾犪,Ｓａ）、肠炎沙门菌（ＳＥ）和鼠伤寒沙门菌（ＳＴ） 的方法。方法　根据沙门菌犪犮犲犃基因、肠炎沙门菌特异序列（ＧｅｎＢａｎｋ：ＡＦ３７０７０７．１）、鼠伤寒沙门菌的ＳＴＭ４５９９ 序列（ＧｅｎＢａｎｋ：ＡＥＲＶ０１００００２３．１）,分别设计引物和ＴａｑＭａｎ探针,在探针的５′端分别标记ＦＡＭ、ＶＩＣ、ｃｙ５,建立基于ＴａｑＭａｎ探针三重实时荧光ＰＣＲ 检测沙门菌的方法。结果　２９ 种不同血清型沙门菌均扩增出犪犮犲犃基因,肠炎沙门菌和鼠伤寒沙门菌的引物和探针分别特异性地扩增出１５株肠炎沙门菌和１１株鼠伤寒沙门菌,而其他血清型沙门菌和１７株非沙门菌扩增结果阴性。犪犮犲犃、肠炎沙门菌、鼠伤寒沙门菌的三重荧光ＰＣＲ 扩增效率分别为８９％、８７％、９０％,最低检测浓度分别达到２８０ｃｆｕ／ｍｌ、２６０ｃｆｕ／ｍｌ、３００ｃｆｕ／ｍｌ。结论　本研究建立的方法特异性好、灵敏度高,可用于食品中沙门菌、肠炎沙门菌和鼠伤寒沙门菌的特异性检测。     

应用聚合酶链反应（PCR）检测乙型肝炎患者的HBV一DNA

应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎＲｅａｃｔｉｏｎ，ＰＣＲ）检测１０８例乙型肝炎患者血清中ＨＢＶ一ＤＮＡ，在不同类型的ＨＢＶ感染标志物（ＨＢＶＭ）的血清中结果有所不同：（１）在ＨＢｓＡｇ、ＨＢｅＡｇ和抗ＨＢｃ阳性血清中，ＰＣＲ阳性率８６．１１％（３１／３６）；（２）在ＨＢｓＡｇ、抗ＨＢｅ和抗ＨＢｃ阳性血清中，ＰＣＲ阳性率５５．８８％（１９／３４）；（３）在抗ＨＢｓ、抗ＨＢｅ和ＨＢｃ阳性血清中，ＰＣＲ阳性率为２０％（２／１０）；（４）在抗ＨＢｅ和抗ＨＢｃ阳性血清中，ＰＣＲ阳性率为２５％（２／８）；文中就ＰＣＲ检测的结果及临床意义作了扼要的讨论。     

生奶中小肠结肠炎耶尔森菌和嗜水气单胞菌的复合和半巢穴PCR检测方法

建立单引物 (ｓｌ)、复合 (ｍ)和半巢穴 (ｓｎ)聚合酶链反应 (ＰＣＲ)法检测生奶样品中小肠结肠炎耶尔森菌和嗜水气单胞菌。设计 2对寡核苷酸引物 ,采用ＰＣＲ方法扩增小肠结肠炎ｙｓｔ基因片段和嗜水气单胞菌Ａｅｒ基因片段 ,其长度分别为 1 4 5ｂｐ和 2 0 9ｂｐ。检测接种的生奶中两种病原体 ,ｓｌＰＣＲ和ｍＰＣＲ方法的检出限约为每毫升 1× 1 0 2 ｃｆｕ(每个ＰＣＲ反应 0 .5ｃｆｕ) ;检测自然感染的生奶 ,ＰＣＲ和培养法小肠结肠炎耶尔森菌的检出率分别为53%和 36 % ,检出嗜水气单胞菌分别为 2 3%和 1 4 %。结果表明 ,直接ＰＣＲ分析…     

嗜肺军团菌环介导等温扩增检测方法的建立及应用简

目的建立一种灵敏、快速、安全、方便适合口岸现场应用的嗜肺军团菌检测方法。方法分析嗜肺军团菌基因组序列,选择特异性基因片段设计引物,建立环介导等温扩增（loop mediated isothermal amplification,LMAP）方法,并优化反应条件。结果建立的方法特异性好,灵敏度可达103cfu/ml,检测能力与荧光定量PCR法相当。结论应用环介导恒温扩增技术建立的嗜肺军团菌检测方法,灵敏度高,特异性好,适合现场快速检测。     

Incidence of Legionella in warm water systems in Saxony-Anhalt

The incidence of Legionella in warm water systems of Sachsen-(Saxony-)Anhalt was investigated. The Legionella were isolated from water samples using plate cultures followed by serotyping methods. In high-risk areas of Legionella infections such as hospitals and homes for the aged, 48% respectively 43% of the samples were positive. Warm water systems of 61% of the hospitals and 43% of homes for aged people were found to be contaminated with Legionella. The number of Legionella were most frequently (50.7%) between 10 and 100 colony-forming units/ml (cfu/ml). High-level Legionella contamination (> 1000 cfu/ml) were detected only in 0.6% of the samples. Legionella pneumophila serogroup 1 (L.p. SG 1) was identified rarely. The reasons for positive Legionella findings are old drinking water heating systems and conduits. To decrease Legionella growth, reconstruction of the old systems according to the recommendations [1, 2] is imperative.     

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.     

Detection of Legionella pneumophila in water samples by quantitative culture and an antigen detection assay

We evaluated the new Legionella pneumophila antigen detection assay Binax Equate for quantitative determination of legionellae in potable water samples. Seventy-seven water samples from different sources were investigated by Binax Equate and quantitative culture. Our culture assay is able to detect 20 to 40 cfu per 100 ml water. The rates of detection of legionellae were 1% (1 of 77) for the antigen detection assay and 25% (19 of 77) by culture. We were able to detect antigen in one water sample with 28 cfu per ml L. pneumophila serogroup 1. In in-vitro experiments the antigen assay had a sensitivity of about 333 cfu per ml when the bacteria were added directly to the test tubes and about 1000 cfu per ml when a simulated water sample was investigated. None of the water samples positive for L. pneumophila serogroup 2 to 14 was positive in the Binax Equate. The new antigen assay proved to be a valuable tool for investigating heavy L. pneumophila Serogroup 1 contamination in potable water systems but lacks sufficient sensitivity to be used in the surveillance of water supplies.     

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。     

PCR方法捕获环境水样中嗜肺军团菌DNA

嗜肺军团菌是军团病的主要病原体，它所导致的军团病病例占临床病例的80％以上，多数军团病暴发由嗜肺军团菌所引起。通过针对嗜肺军团菌种保守基因（mipgene）设计的引物，我们建立了一种多聚酶链反应（PCR）检测方法，可以准确捕获环境标本中的嗜肺军团菌，同时具有很好的特异性，其最低可检出18cfu／ml的细菌。该方法的建立为嗜肺军团菌病的诊断以及在军团病流行病学调查中嗜肺军团菌病原的追踪提供了一条便捷、准确、可靠的途径。     

A patient with Legionella pneumophila serogroup-3 pneumonia, detected by PCR

A 59-year-old man was hospitalised because of dyspnoea, productive cough, fever, chills and malaise. Severe community-acquired pneumonia was diagnosed. Legionella urinary antigen testing, which can only detect serogroup 1, and the first culture ofa bronchoalveolar lavage (BAL) fluid sample were negative for Legionella. However, L. pneumophila DNA was detected by PCR in the BAL washing sample. Eventually, L. pneumophila serogroup 3 was isolated from this specimen by repeated culture. Although, in The Netherlands, legionellosis is caused by L. pneumophila serogroup 1 in more than 90% of all cases, this case demonstrates that a negative result of urinary antigen testing does not necessarily exclude this diagnosis. It is therefore advocated to expand the diagnostics to a Legionella PCR on respiratory material of patients with clinical signs of Legionella pneumonia in whom the urinary antigen test is negative.     

Legionella contamination of hospital water supplies: monitoring of private healthcare facilities in Bologna, Italy

The hot water supplies of 11 private healthcare facilities in the city of Bologna, Italy, were monitored for the presence of Legionella spp. Four samplings were made in each establishment over a period of one year and in total 121 samples were collected from distribution points situated near the water-boiler and inside the wards (taps and showers). Legionellae were recovered from all the water supplies in question: Legionella spp. in 86.8% of samples, L. pneumophila in 82.6% of samples. L. pneumophila was found in all the water supplies at levels averaging above 10(4)cfu/L in five health facilities and reaching a maximum concentration of 10(7)cfu/L. The only parameter to have affected the presence of legionellae was the water temperature, which was seen to be inversely correlated to the concentration of Legionella spp. Despite the high levels of contamination from L. pneumophila, no cases of nosocomial Legionnaires' disease were reported during the period of the study.     

Comparison of the ActiDes-Blue and CARELA HYDRO-DES technology for the sanitation of contaminated cooling water systems in dental units

Background: The hygienic-microbiological control of 6 dental units being in use for the past 16 years revealed a significantly increased microbial contamination of their cooling water system. In order to comply with the requirements of the drinking water directive ("Trinkwasserverordnung"), the commercially available production system ActiDes, producing on-site ActiDes-Blue which is based on hypochlorous acid (HOCl) and generated by anodic oxidation, was investigated. Method: Water samples from the 6 contaminated dental units were examined for the total number of colony forming units (cfu), contamination with molds, L. pneumophila and P. aeruginosa. The control period for the total colony count was 4 weeks (8 samples/unit). The subsequent application phase of the ActiDes-Blue procedure was 6 months (31 samples/unit). Additionally, the redox potential and the pH value were measured.Futhermore, the decontamination agent CARELA HYDRO-DES, a two component agent based on H(2)O(2) with the addition of a mixture of sodium hydrogen sulphate and sulphuric acid in an aqueous solution effective at 0.1% and higher, was applied in a unit that had been put out of service for a month before. Before application, the system was first filled with a 5% solution of the alkaline pre-cleaning agent CARELA Solvent for bacterial slime; the system was left with this solution for 1 h. The pre-cleaning agent was then completely displaced from the system with tap water and a decontaminating solution of 5% CARELA HYDRO-DES and left in place for 1 h.Results: Drinking water quality level was reached only twice during the control phase. The average values of the dental units ranged between 3,633 CFU/ml and 29,417 c/ml. During the application phase, drinking water level could be achieved in 11 water samples. In another 6 water samples a total colony count of <150 cfu/ml was reached. The average values for the dental units' total colony count ranged between 529 cfu/ml and 87,450 cfu/ml. No significant differences between the control phase and the action phase could be demonstrated. During the control phase, contamination of the water samples with a mold was noticed so that examinations for molds were carried out beyond the scope of the drinking water directive. For this parameter as well, no significant differences between the phases of the study could be shown.The Legionella load of the dental units was low. L. pneumophila were yielded in only 4 out of 130 water samples. During the control phase, twice colony counts at 50 cfu/1,000 ml and 110 cfu/1,000 ml were measured. During the action phase, counts with Legionella spp. could be measured at 5 cfu/1,000 ml for one unit only. Also, with 1-10 cfu/100 ml, the P. aeruginosa contamination was low. During the application phase, it ranged between 0-7 cfu/100 ml.Redox potential and pH value showed a slight decrease during the application phase.Before treatment with CARELA Solvent and CARELA HYDRO-DES, the initial contamination of the total count of bacterial colonies was 1,432 cfu/ml at 22°C and 846 cfu/ml at 36°C as well as >1,000 cfu/100 ml for molds. 1 h after the decontamination, no bacteria and molds could be detected in 1,000 ml of tap water. Despite the fact that the unit was not used any longer, after 7 d the bacterial colony count was 3 cfu/ml at 22°C and 2 cfu/ml at 36°C while molds could not be detected. Even after a rest time of 14 d only 167 cfu/ml or 42 cfu/ml could be yielded. Molds were further not cultivable. A material damage could not be observed. Discussion: Pertaining to the ActiDes technology's effectiveness, it has to be pointed out that the dental units investigated were those used for dental students' teaching and therefore were clearly less frequently used than clinically used units in a dental practice. This resulted in distinctly longer stagnation periods which favored formation of biofilms. Conclusions: In summary, the ActiDes technology and ActiDes-Blue showed not to be sufficiently effective for the sanitation of contaminated water reservoirs in dental units under aggravated conditions of repeated and longer periods of non-use in connection with longer water stagnation periods. In comparison, the biofilm was sustainably eliminated through the combined application of CARELA(?) Solvent for Bacterial Slime with subsequent decontamination using CARELA(?) HYDRO-DES.