Frankia的REP-PCR、RAPD-PCR鉴定及遗传多样性研究

本文采用溶菌酶法、CTAB法、微波法及CTAB+微波法等4种方法进行Frankia菌Cp11基因组DNA的提取。结果表明四种方式均可行，但以CTAB法及微波法最为有效。提取方法与REP-PCR带型间关系的研究表明，方法上的差异不会影响到REP-PCR带型的变化。对14株Frankia菌株作REP-PCR分析，并对之进行比较分类，证明REP-PCR方法不但能实现菌株间的差异鉴别，还能有效地进行Frankia菌的分类。这一结果与DNA同源相关性所得的分类结果具有良好的相关性。选用20个随机引物，对分自2个分类接种群的8株Frankia纯培养的总DNA进行随机扩增。其中引物OPW15和OPW16能扩增得到较为稳定的RAPD图谱。扩增产物分子量大都分布在0.5-4Kb之间。从稳定的RAPD扩增图谱看，Frankia菌间存在有丰富的遗传多样性；在选定适当引物情况下，能依据共同带型将Frankia菌化归为同一分类接种群。

rep-PCR对赤杨根瘤内局Frankia菌基因多样性研究

本文应用重复基因外回纹序列rep-PCR方法直接扩增长日山三种不同赤杨和云南不同地区早冬瓜根瘤内的Frankia菌基因，所获得的REP指纹图谱揭示Frankia菌具有丰富的基因多样性。研究结果表明Frankia菌多样性随地域，海拔，坡问不同而变：多样性指数与自然选抒）玉力大小成正相关：自然选择压力是产生和保持Frankia菌基因多样性的重要因子。同时发现Frankia菌基因型与地域有紧密关系：所观察的云南各地区中，感染高黎贡山旱冬瓜的Frankia菌基因多样性最为丰富。推测与高黎贡山旱冬瓜共生的Frankia可能作为种储备库，为其它地区的寄主植物提供了祖先菌株。在对长白山三种赤杨（东北赤杨，西伯利亚赤杨和色赤杨）根瘤内几百游殆菌基因多样性考察过程中发现，Frankia菌基因类型与寄主赤杨种型关系密切。同时海拔对感染东北赤杨的Frankia菌基因多样性也有一定影响。感染西伯利亚赤杨和色赤杨的Frankia菌享有相同的基因型，可能有较近的亲缘关系。

利用rep-PCR研究云南尼泊尔桤木根瘤内Frankia基因多样性

利用rep-PCR指纹技术对云南5个地区的尼泊尔桤木根瘤内Frankia菌基因多样性进行研究,实验发现Frankia菌呈现丰富的基因多样性.所有71个样品被分为11种不同的基因类型.除高黎贡山外,不同地域都有某种基因型占优势,显示Frankia菌基因型与地域有紧密关系.所观察的5个地区中,高黎贡山Frankia菌基因型种类最多,多样性指数最高,基因多样性最为丰富.结合高黎贡山地理资料及其它生物多样性相关研究,推测与高黎贡山尼泊尔桤木共生的Frankia可能作为种储备库,为其它地区的寄主植物提供了祖先菌株.图8表1参13

Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Strain variation amongst clinical and potable water isolates of M.kansasii using automated repetitive unit PCR

Mycobacterium kansasii is a pulmonary pathogen that has been grown readily from municipal water, but rarely isolated from natural waters. A definitive link between water exposure and disease has not been demonstrated and the environmental niche for this organism is poorly understood. Strain typing of clinical isolates has revealed seven subtypes with Type 1 being highly clonal and responsible for most infections worldwide. The prevalence of other subtypes varies geographically. In this study 49 water isolates are compared with 72 patient isolates from the same geographical area (Brisbane, Australia), using automated repetitive unit PCR (Diversilab) and ITS RFLP. The clonality of the dominant clinical strain type is again demonstrated but with rep-PCR, strain variation within this group is evident comparable with other reported methods. There is significant heterogeneity of water isolates and very few are similar or related to the clinical isolates. This suggests that if water or aerosol transmission is the mode of infection, then point source contamination likely occurs from an alternative environmental source.

长白山三种赤杨根瘤内Frankia基因多样性的REP分析

利用REP PCR方法研究了长白山不同海拔处 3种赤杨根瘤内Frankia菌基因多样性。结果表明 ,赤杨根瘤内Frankia菌存在丰富的基因多样性。宿主和海拔因素共同影响长白山赤杨属Frankia菌的基因多样性 :东北赤杨Frankia菌的宿主专一性很强 ,西伯利亚赤杨和色赤杨的Frankia菌的有较近的亲缘关系。不同海拔的同种宿主共生的Frankia菌基因类型不同 ,相同海拔的西伯利亚赤杨和色赤杨的Frankia菌出现宿主混乱现象

用REP-PCRDNA指纹鉴别Frankia菌

用4种方法提取CPI1菌基因组DNA，并进行了REP-PCR扩增，结果表明，提取方法不影响REP-PCR带型。对14株Frankia菌株作REP-PCRDNA指纹分析，并对之进行比较鉴别，证明REP-PCR指纹法能有效地实现菌株间的差异鉴别，其结果与DNA同源相关性所得结果具有良好的相关性。

Tipificação molecular de amostras de Brucella spp. utilização a técnica de BOX-PCR

O gênero Brucella é formado por coco-bacilos Gram negativos patogênicos ao homem e animais, sendo classificado como patógeno de grupo de risco III. A identificação dessas bactérias apresenta várias limitações como: exigência de inoculação em vários meios, tempo de incubação longo e necessidade de soros imunes e bacteriófagos. Devido à sua alta patogenicidade e ao longo tempo de exposição dos laboratoristas à bactéria, a brucelose é uma das infecções mais freqüentemente adquiridas em laboratório. Além da contaminação em laboratório, a transmissão ao homem pode ocorrer através de animais infectados e ingestão de produtos derivados, como o leite cru. A procura de métodos rápidos de identificação das espécies e biovares pode ser útil para diminuir os riscos do manuseio desta bactéria e na tomada de medidas de controle epidemiológico. O principal objetivo deste trabalho foi facilitar a classificação de cepas de referência de Brucella spp. e isoladas no Brasil utilizando a técnica de rep-PCR com oligonucleotídeos do elemento BOX, uma seqüência repetida presente no genoma de várias bactérias. Foram analisados 38 isolados representando diferentes espécies e biovares de Brucella sp. e 13 isolados de gêneros relacionados como controle da especificidade da reação. Foi realizada uma confirmação prévia dos isolados de brucela por testes bioquímicos e PCR gênero-específica. A técnica de BOX-PCR agrupou todas as espécies e biovares de Brucella em um único grupo com nível de similaridade entre 100 e 74%. Diferenças entre os isolados, quanto a presença ou ausência de bandas, puderam ser observadas. Entretanto, essas divergências não caracterizam uma espécie ou biovar. Bandas comuns a todos os isolados de Brucella sp. podem caracterizar o gênero.

Rapid genotyping of Pseudomonas aeruginosa isolates harboured by adult and paediatric patients with cystic fibrosis using repetitive-element-based PCR assays

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38% of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam, The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P, multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry. (C) 2000 Elsevier Science B.V. All rights reserved.

Characteristics of nontuberculous mycobacteria from a municipal water distribution system and their relevance to human infections

This thesis documented pathogenic species of nontuberculous mycobacteria in the Brisbane water distribution system. When water and shower aerosol strains were compared with human strains of mycobacteria, the study found that the likelihood of acquiring infection from municipal water was specific for four main species. The method for isolation of mycobacteria from water was refined, followed by sampling from 220 sites across Brisbane. A variety of species (incl 15 pathogens) were identified and genotypically compared to human strains. For M. abscessus and M. lentiflavum, water strains clustered with human strains. Pathogenic strains of M. kansasii were found, though non-pathogenic strains dominated. Waterborne strains of M. fortuitum differed to human strains. Extensive home sampling of 20 patients with NTM disease, supported the theory that the risk of acquiring NTM from water or shower aerosols appears species specific for M. avium, M. kansasii, M. lentiflavum and M. abscessus.

Mycobacterium abscessus isolated from municipal water : a potential source of human infection

Background Mycobacterium abscessus is a rapidly growing mycobacterium responsible for progressive pulmonary disease, soft tissue and wound infections. The incidence of disease due to M. abscessus has been increasing in Queensland. In a study of Brisbane drinking water, M. abscessus was isolated from ten different locations. The aim of this study was to compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods Between 2007 and 2009, eleven isolates confirmed as M. abscessus were recovered from potable water, one strain was isolated from a rainwater tank and another from a swimming pool and two from domestic taps. Seventy-four clinical isolates referred during the same time period were available for comparison using rep-PCR strain typing (Diversilab). Results The drinking water isolates formed two clusters with ≥97% genetic similarity (Water patterns 1 and 2). The tankwater isolate (WP4), one municipal water isolate (WP3) and the pool isolate (WP5) were distinctly different. Patient isolates formed clusters with all of the water isolates except for WP3. Further patient isolates were unrelated to the water isolates. Conclusion The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same geographical area, strengthens the possibility that drinking water may be the source of infection in these patients.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes - adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

污灌和农药对农田土壤假单胞菌种群的影响

随着环境污染问题的日益严重，微生物修复起到越来越重要的作用。假单胞菌是土壤微生物中最重要、研究最多的细菌之一，能降解简单和复杂的有机物，它们因此而广泛的存在于土壤和水体。但有关于石油、重金属及农药污染物对农田土壤假单胞菌多样性及种群结构的影响却缺乏全面和系统的认识。 本论文首次采用传统微生物培养方法与PCR-DGGE等现代微生物分子生态学研究方法相结合的手段，系统评价了长期含石油和重金属污水灌溉对中国最大的石油、重金属污灌区——沈抚、张士灌区农田土壤中的假单胞菌多样性及其种群结构的影响。同时，本论文也研究了乙草胺、甲胺磷对黑土假单胞菌多样性及种群结构的影响。得出以下结果： 石油污灌区土壤中总的假单胞菌多样性显著高于重金属污灌区；石油污灌区旱田土壤假单胞菌多样性接近于对照清洁土壤,同时低于相似污染程度的石油污灌区水田土壤。进一步测序发现，Pseudomonas mendocina、Pseudomonas stutzeri、Pseudomonas aeruginosa是所分析石油和重金属污灌区土壤中的优势类群，说明在长期污染胁迫下这3种假单胞菌分别得到了不同程度的富集。DGGE 结果显示石油和重金属污染土壤样品的可培养假单胞菌多样性没有显著差异，但均低于对照清洁土壤样品。对各个土壤样品可培养假单胞菌菌株进行REP-PCR基因分型，结果表明这些假单胞菌之间有显著的遗传差异。进一步测序表明，土壤样品中可培养假单胞菌优势类群中含有Pseudomonas. fluorescens 和Pseudomonas. Putida两种。 黑土农田土壤中使用乙草胺会严重降低总的及可培养假单胞菌群落的多样性，而且在5周内不能恢复。而甲胺磷处理土壤与对照相比则差异不显著，并且经过一段时间的适应，土壤中的总的及可培养假单胞菌种群不仅得到恢复而且超过对照。对各处理土壤总的及可培养假单胞菌DGGE谱带类型聚类分析，发现乙草胺、甲胺磷处理土壤样品均各自聚为一簇，说明农药污染类型是影响土壤中假单胞菌种群结构的重要因素。