应用PCR技术检测媒介恙螨体内恙虫病立克次体(英语)

本文PCR技术的引物是参考恙虫病立克次体Karp株Sta 58序列设计合成的一对DNA引物.以恙虫病立克次体DNA为模板,成功扩增长约1kb的DNA片段.该对引物首次应用于人工成虫腹内接种羔虫病立克次体(人工接种法)和幼虫叮咬恙虫病鼠(叮咬病鼠法)的我国主要恙虫病媒介地里纤羔螨的研究.PCR检测人工接种法成虫的不同时期和经卵传递的子代立克次体DNA均获满意结果,立克次体在恙螨体内生长持续360天及产卵孵出的第4代幼虫均呈阳性.叮咬病鼠法立克次体在恙螨亲代饱食蚴、若蛹、若虫、成虫及第2代子产代幼虫,PCR检测均呈阳性.结果显示PCR技术检测恙螨体内恙虫病立克次体的特异性和敏感性高;体外基因扩增检测恙螨体内及鼠宿主体内立克次体是流行病学调查的新的敏感方法.本法为恙虫病分子体流行病学调查提供了新的应用技术,亦为临床病人诊断提供了敏感方法.     

Consistency of the Key Genotypes of Orientia tsutsugamushi in Scrub Typhus Patients, Rodents, and Chiggers from a New Endemic Focus of Northern China

Scrub typhus is one of the most common infectious diseases of rural south and southeastern Asia and the western Pacific. It emerged in Shandong Province in northern China from autumn to winter of 1986. Since then, the “autumn–winter type scrub typhus” has been found in many areas of northern China. However, the principle genotypes of Orientia tsutsugamushi still remain unknown. This study was undertaken to identify the genotypes of O. tsutsugamushi obtained from scrub typhus patients, chigger mites and rodents from the focal point of the problem in Shandong Province. Forty-four isolates from patients, rodents, and chiggers, 47 blood clots from patients during the acute phase, 10 eschars from patients during the convalescence phase and 16 pools of larval chiggers were tested for the scrub typhus antigen 56-kD protein (Sta56) gene by nested PCR methodology and additional sequence analysis including DNA sequence alignment and phylogenetic analysis. Based on nested PCR, ninety-five initial PCR-positive samples produced amplicons using Kawasaki strain-specific primers, while the other two (the FXS4 and LHGM2 strains) produced amplicons using Karp strain-specific primers. The partial Sta56 gene sequence analysis indicated that the sequence homologies of 3 selected isolates (the B16, FXS2, and XDM2 strains) and 7 eschars out of the 95 samples, which were nested PCR-positive using Kawasaki strain-specific primers, were 94–98 % to that of Kawasaki strain. The sequence homology of the FXS4 and LHGM2 strains to that of the Karp strain was respectively 83 and 96 %. These findings implied that the key genotypes of O. tsutsugamushi in patients, rodents, and chiggers in Shandong Province were identical and similar to Kawasaki strains.     

Dual exposure of Rickettsia typhi and Orientia tsutsugamushi in the field‐collected Rattus rodents from Thailand

Field‐collected rodents and fleas from ten provinces covering four regions of Thailand were investigated for possible rickettsial pathogen infections. The 257 trapped‐rodents belonged to 12 species. Five species of Genus Rattus accounted for 93% of the total capture, of which Rattus exulans and Rattus norvegicus were the two major species caught. All flea specimens, removed from trapped rodents, were identified as Xenopsylla cheopis. The PCR technique was performed on ectoparasite specimens to detect the presence of murine typhus pathogen (Rickettsia typhi) and scrub typhus pathogen (Orientia tsutsugamushi). Thirteen flea specimens (2.6 %) were found to be positive for R. typhi but none for O. tsutsugamushi. An ELISA technique was used to detect the rodent's antibodies against R. typhi and O. tsutsugamushi. Sixty‐one rodent serum samples (23.7%) were positive for R. typhi specific IgM, IgG, or both, while 47 of the samples (18.3%) were positive for O. tsutsugamushi. Twenty serum samples from R. norvegicus (7.8%) had detectable antibodies against both R. typhi and O. tsutsugamushi. Our findings revealed the existence of the dual infection of rickettsial pathogens in the same natural hosts.     

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37^oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39^oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.     

Metabolic Responses to Orientia tsutsugamushi Infection in a Mouse Model

Tsutsugamushi disease is an infectious disease transmitted to humans through the bite of the Orientia tsutsugamushi-infected chigger mite; however, host-pathogen interactions and the precise mechanisms of damage in O. tsutsugamushi infections have not been fully elucidated. Here, we analyzed the global metabolic effects of O. tsutsugamushi infection on the host using ¹H-NMR and UPLC-Q-TOF mass spectroscopy coupled with multivariate statistical analysis. In addition, the effect of O. tsutsugamushi infection on metabolite concentrations over time was analyzed by two-way ANOVAs. Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed distinct metabolic patterns between control and O. tsutsugamushi-infected mice in liver, spleen, and serum samples. O. tsutsugamushi infection caused decreased energy production and deficiencies in both remethylation sources and glutathione. In addition, O. tsutsugamushi infection accelerated uncommon energy production pathways (i.e., excess fatty acid and protein oxidation) in host body. Infection resulted in an enlarged spleen with distinct phospholipid and amino acid characteristics. This study suggests that metabolite profiling of multiple organ tissues and serum could provide insight into global metabolic changes and mechanisms of pathology in O. tsutsugamushi-infected hosts.     

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR

Aims: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species‐specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. Methods and Results: The nucleotide sequences of 24 Vibrio and seven non‐Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510‐bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. Conclusions: The groEL gene is a potential marker for the species‐specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. Significance and Impact of the Study: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.     

Morphological and molecular identification of some closely related Pythium species in Egypt

Twelve isolates of Pythium species (P. aphanidermatum, P. deliense, P. ultimum var. ultimum and P. ultimum var. sporangiiferum) from different hosts were compared from morphological, pathological and molecular viewpoints. Minimum, optimum and maximum temperatures of P. aphanidermatum and P. deliense were similar while those of P. ultimum var. ultimum and P. ultimum var. sporangiiferum were also similar. All tested isolates were highly virulent against cucumber seedlings with 100% damping-off. RAPD data using three different primers revealed that strains of P. ultimum var. ultimum and P. ultimum var. sporangiiferum are distinct from each other. This data can be used to separate those species from P. aphanidermatum and P. deliense. In contrast, RAPD data cannot be used to separate P. aphanidermatum and P. deliense. Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among the tested isolates.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. I. Inhibition of Growth of Rickettsia tsutsugamushi,Karp Strain,in Cultured Murine Embryonic Cells and Macrophages by Recombinant Tumor Necrosis Factor-Alpha

Recombinant murine tumor necrosis factor-alpha (TNF-α) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF-α was also evident when peritoneal exudate macrophages or bone marrow-derived macrophages were used as the host cell for rickettsial growth. Interferon-gamma (IFN-γ), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow-derived macrophages. The effect of TNF-α on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN-γ. Treatment of the cell line with TNF-α delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN-γ-mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF-α may inhibit rickettsial growth in cells other than macrophages.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Effect of European wheat striate mosaic, acquired transovarially, on the biology of its planthopper vector Javesella pellucida

The proportion of infective nymphs of Javesella pellucida in the progenies of female vectors of European wheat striate mosaic (EWSM) disease ranged from 85 to 96%; 71% of these nymphs infected plants within one week of hatching. Inbreeding for one or two generations significantly decreased the viability of J. pellucida eggs, but EWSM had no effect on the viability of eggs laid by inbred or outbred vector lines. However, EWSM acquired transovarially usually increased the mortality of J. pellucida nymphs by 13 to 17%, although mortality was as high as 30% in some vector lines. EWSM, acquired transovarially for two generations, decreased the longevity of adult males and females of J. pellucida by 14%. Inbreeding for two generations resulted in 40% increase in the mortality of nymphs and more than 50% reduction in the longevity of adults.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor-Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.     

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real‐time PCR targeting the 16S ribosomal RNA gene, serotype‐specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

Molecular evidence of occurrence of Tomato Leaf Curl New Delhi Virus infecting cucurbits in several states in India

Abstract

Tomato Leaf Curl New Delhi Virus (ToLCNDV) is an important begomovirus constraint to the production of cucurbitaceous and other crops in India and its subcontinent. In this study first time, symptomatic samples of different cucurbits were collected from Telangana, Uttar Pradesh (Varanasi) and Madhya Pradesh location in India. The symptomatic samples were associated with begomovirus infection was confirmed by PCR amplification using specific universal primers Deng 540/541. Thirteen out of fifteen samples were amplified with newly designed bipartite specific primers JKR 58?F and JKR 59?R. In eleven out of thirteen bipartite samples contain betasatellite, confirmed by PCR amplification using specific primer CLB36F and CLB37R. The amplified PCR product with JKR58F and 59R of bitter gourd sample collected from Telangana was sequenced. The sequence was share 97.53% similarity with ToLCNDV infecting bottle gourd in Haryana in India (FN645905). Phylogenic analysis revealed that ToLCNDV infecting cucurbits are different, from ToLCNDV infecting tomato.     

Life cycle,epizootiology, and horizontal transmission of Amblyospora (Microspora: Amblyosporidae) in a univoltine mosquito, Aedes stimulans

Amblyospora infections in Aedes stimulans are transovarially transmitted by females infected in the previous year. Pathogen development in progeny is dimorphic and host sex dependent. In males, the pathogen invades fat body tissue and undergoes an extensive developmental sequence which kills the host and results in the formation of eight haploid spores enclosed in an accessory membrane that are not infectious to other larvae. In females, the pathogen invades host oenocytes and undergoes a simple developmental sequence which has no detrimental affect on longevity, fecundity, oviposition, or egg hatch, and results in the formation of binucleated spores that infect the ovaries and ensure transmission to the next generation. Transovarial transmission is continuous and is the major way in which these microsporidia are maintained from year to year, but is incapable of maintaining infections in breeding populations because of low transmission rates and is not sufficient to account for the types and levels of infection observed in the field. Horizontal transmission is reported for the first time. It occurs sporadically during the early stages of larval subsequently disseminated to oenocytes of adult hosts and are transovarially transmitted by females to filial host generations. This pathway of transmission provides the necessary mechanism whereby these microsporidia can reenter the mosquito population and thus perpetuate themselves.     

昆虫共生菌的垂直传播

共生菌普遍存在于昆虫体内,它们能够为宿主昆虫提供生长发育所必需的氨基酸、固醇类等营养物质,还能提高昆虫适应高温、寄生虫、病毒等不利环境因素的能力,昆虫则为共生菌提供稳定的生存环境和营养物质,昆虫与共生菌相互依存。多数情况下,共生菌通过垂直传播在宿主代次间进行传播,即共生菌由母代传递给子代。结合最近几年相关研究,本文综述了不同昆虫共生菌的垂直传播模式。除极少数肠道共生菌通过污染卵壳被宿主幼虫取食得以垂直传播外,垂直传播的共生菌多为经卵传播。根据侵染时期的不同,共生菌经卵传播模式多数可分为以下4种：侵染宿主昆虫幼虫中的生殖干细胞、侵染宿主昆虫年轻雌成虫中的生殖干细胞、侵染宿主昆虫雌成虫中的成熟卵母细胞以及侵染宿主昆虫囊胚期胚胎。其中,有些共生菌是以共生菌菌胞整体侵染的方式进入到宿主卵巢。另外,少数肠道共生菌也通过卵巢进行垂直传播,此类共生菌先侵染卵巢侧输卵管并在侧输卵管聚集,待卵排放至侧输卵管时再进入到卵中。在文中,我们也探讨了昆虫共生菌垂直传播过程中的细胞机制和免疫机制,包括共生菌避开宿主免疫反应、共生菌通过内吞作用进入卵巢以及不同共生菌间的协同作用等。     

环境样本中微型和微微型浮游植物高通量测序的引物优化

分别以18Sr DNA的V4区和V9区为目标基因,采用高通量测序平台和生物信息学方法,分析海水样品中微型和微微型浮游植物多样性。利用在线分析软件对V4(F/R)、V9(F/R)和C4(F/R)3对引物的敏感性、特异性进行了评估和比较,发现自行设计的引物V4(F/R)对真核藻类的扩增特异性高于V9(F/R)和C4(F/R)。高通量测序结果显示,检测样品平均获得68834条原始序列,高质量数据占99%以上,获得基因注释的序列数达94%以上。3对引物V4(F/R)、V9(F/R)、C4(F/R)鉴定的平均微型/微微型浮游植物OTUs数分别为78、42、58,引物V4(F/R)鉴定效率相对较高,同时对细小微胞藻(Micromonas pusilla)、(金牛微球藻Ostreococcus tauri)、密球藻(Pycnococcus provasolii)、抑食金球藻(Aureococcus anophagefferens)、赤潮异弯藻(Heterosigma akashiwo)等优势种检出频率高于引物V9(F/R)。相对已发表的2对引物,设计的引物V4(F/R)对海洋微型和微微型藻类多样性检测更为高效。     

Specific PCR assays for the detection of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> causing green mold disease during mushroom cultivation

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50 fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.     

Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.