分子生物学方法在食品微生物检测中的应用

近年来,食品安全受到广泛关注。及时、准确地检测出食品中的病原微生物是食品安全检测的重要内容,食品病原微生物的分子生物学检测技术因其特异性和灵敏性而备受瞩目。我们主要介绍了基因探针检测法、PCR检测法和基因芯片检测法的原理、开发及其在食品微生物检测中的应用。     

森林生态系统中土壤微生物的作用与应用

土壤微生物作为森林生态系统的重要组成部分,在林业可持续发展中扮演重要角色．本文就土壤微生物在森林生态系统中的作用作了综述．内容包括：土壤微生物区系及生物量在林业可持续发展中的作用、菌根在林业可持续发展中的作用、植物病原微生物对林业可持续发展的影响、PGPR在林业可持续发展中的作用．并对其今后的发展趋势及应用前景等问题作了探讨,提出了今后应进一步开展的研究方向．     

人工湿地与环境卫生安全

人工湿地是国内外应用较为广泛的一种污水处理技术。近年来,人工湿地的环境卫生安全问题越来越受到人们的关注。人工湿地中病原微生物的去除或失活受到诸多因素的影响,其过程和机制与传统的二级污水处理工艺有较大区别,选择适宜的指示微生物和病原微生物并研究其行为是进行人工湿地环境卫生安全评价的关键。本文论述了病原微生物在人工湿地中的归宿以及人工湿地可能对环境卫生安全造成的影响,综述了国内外的研究现状,指出了该方面研究的必要性和迫切性。     

水中病原微生物分子检测技术研究进展

基于PCR方法的多种分子检测技术已广泛的应用于水体病原微生物的检测中。而以DNA芯片为代表的微型化、快速化手段将是未来检测技术的发展方向,可实现对病原微生物实时和快速的检测。新检测技术的发展有利于建立水体污染早期预警机制,同时,可靠的病原微生物检测方法可降低有害微生物对人类健康的影响。对水体病原微生物分子检测方法及其在水污染相关疾病风险控制中所扮演的重要角色进行阐述。     

Src和Abl酪氨酸蛋白激酶家族参与病原微生物感染的研究进展

Src和Abl家族激酶属于非受体型酪氨酸激酶(Nonreceptor tyrosine kinase,NRTK)家族重要成员,广泛存在于各种细胞中,参与细胞内信号传递并调节细胞生理过程,它们在维持细胞、组织和器官稳态功能中发挥着至关重要的作用。研究表明,Src和Abl家族激酶通过多种机制参与病原微生物的感染(如与病原微生物的脯氨酸基序-PXXP互作)。因此,从Src和Abl家族激酶角度出发探究病原微生物感染机制逐渐成为一个热点。本文就Src和Abl家族激酶的结构特点以及参与病原微生物感染的研究报道进行综述,以期为病原微生物感染的致病机制、防控和药物研发提供参考。     

病原性因子和DNA诊断总论

<正>为了有效地治疗传染病,有必要对引起炎症的病原微生物进行分离、鉴定以及正确的诊断。但是,就整个分离、鉴定过程而言,需要较长时间。为了达到早期诊断的目的,目前正在开发各种快速的诊断方法。在快速诊断方法中,有对存在于检样中的病原微生物的特定抗原进行免疫学检测的方法和对存在于患者血清中的病原微生物的特异性抗体进行检测的方法等。近年来随着分子生物学手段的逐步普及,DNA诊断法已被各个领域广泛采用,现就DNA诊断法与其它方法进行比较:     

《昆虫的寄生性天敌和病原微生物》

全书分二卷。第一卷：寄生性天敌;第二卷：病原微生物。论述了昆虫与寄生性天敌和病原微生物的相互作用和影响。从基础生物学、生物化学和分子生物学的角度进行研究。第一卷：寄生性天敌（V．1Parasites）。共14章,364页。分1．昆虫寄生性天敌发育的模式;2寄生性天敌的作用：在寄生性天敌和寄主相互关系中蛋白质和肽所起的作用;3．昆虫内寄生性天敌和寄主相互关系中激素的作用;4．昆虫寄生性天敌和病原微生物引起的繁殖干扰;5．昆虫寄生性天敌和病原微生物对寄主行为的影响;6．寄生新陈代谢的变化对奇生性天敌营养的影响;7．畸形细…     

浅析分子生物学技术在临床常见病原微生物快速检测中的应用

目的:分析与探讨分子生物学技术在常见病原微生物的快速检测中的应用与作用。方法:将2014年10月至2015年10月期间,我院在临床常见病原微生物的快速检测中所应用的分子生物学技术作为本篇论文的分析对象,回顾性分析相关资料。结果:我院所应用的分子生物学技术主要有三种,即聚合酶链反应(PCR)技术、基因(DNA)芯片技术以及生物传感器技术,上述三种新兴的分子生物学技术都具有自身独特的优点,均可以为临床常见病原微生物的快速检测提供一定的便利条件。结论:在临床常见病原微生物的快速检测中,分子生物学技术发挥着非常大的作用,合理、有效地应用分子生物学技术,具有重要的意义。     

Th1/Th2与PCT检测在感染性疾病诊断中的临床应用

病原微生物引起的感染性疾病是临床中极为常见的一类疾病,此类疾病的预后主要取决于感染的病原微生物类型及疾病的严重程度。早期诊断和积极干预可在很大程度上改善感染性疾病患者的预后。因此,快速、有效地识别感染的病原微生物并对其进行靶向治疗,避免抗生素的滥用显得尤为重要。Th1/Th2在感染性疾病中的作用越来越得到重视,而降钙素原(PCT)是临床感染性疾病诊断和鉴别诊断的早期指标,且具有高度的敏感性和特异性。Th1/Th2和PCT的联合检测对感染性疾病的诊断、病情评估和疗效监测都具有重要的意义。     

现代分子生物学技术在食品、药品微生物检测中的应用

食品与药品中存在的病原微生物直接关系到食品、药品的质量和安全,因此对食品和药品进行微生物检测对保护人类的身体健康具有非常重要的意义。近年来,随着现代分子生物学技术的飞速发展,其在食品和药品的微生物检测中得到了非常广泛的应用。本文主要通过对现代分子生物学技术在食品、药品微生物检测中的应用及进展进行相应的探讨,旨在为新型微生物检测及分型方法的发展提供依据。     

荧光原位杂交技术及其在微生物生态学中的应用

综述了荧光原位杂交技术 (fluorescence in situ hybridization FISH)在微生物生态学领域的各种应用 ,同时就其发展过程、原理及种类做了介绍     

Application of FISH technology for microbiological analysis: current state and prospects

In order to identify and quantify the microorganisms present in a certain ecosystem, it has become necessary to develop molecular methods avoiding cultivation, which allows to characterize only the countable part of the microorganisms in the sample, therefore losing the information related to the microbial component which presents a vitality condition, although it cannot duplicate in culture medium. In this context, one of the most used techniques is fluorescence in situ hybridization (FISH) with ribosomal RNA targeted oligonucleotide probes. Owing to its speed and sensitivity, this technique is considered a powerful tool for phylogenetic, ecological, diagnostic and environmental studies in microbiology. Through the use of species-specific probes, it is possible to identify different microorganisms in complex microbial communities, thus providing a solid support to the understanding of inter-species interaction. The knowledge of the composition and distribution of microorganisms in natural habitats can be interesting for ecological reasons in microbial ecology, and for safety and technological aspects in food microbiology. Methodological aspects, use of different probes and applications of FISH to microbial ecosystems are presented in this review.     

FISH methods in phycology: Phototrophic biofilm and phytoplankton applications

Abstract

Many photosynthetic microorganisms, living attached to immersed substrates or free in the water column, lack distinct morphological details, are small in size and often unculturable. Thus, whole-cell fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a valuable and widely used technique to identify bacteria and protists within their natural communities. FISH methods not only allow direct, cultivation independent determination of community composition, but provide spatio-temporal quantification of microorganisms in the environment. Coupling of FISH techniques to Confocal Laser Scanning Microscopy (CLSM) has become essential for the assessment of diversity and structural integrity in three-dimensional complex biofilm samples. Combining FISH with microautoradiography (FISH-MAR) and microsensors also opens new perspectives in microbial ecology by providing new tools for revealing physiological properties of organisms with single-cell resolution. This paper briefly summarizes the application of FISH methods to phototrophic biofilm and phytoplankton research. The potential of DNA microarray technology in phycological research is highlighted, especially for the fast and accurate identification of HAB (Harmful Algal Bloom) species in marine phytoplankton. Some CLSM and FISH data from phototrophic biofilms from an Italian wastewater treatment plant are shown.     

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.     

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

Molecular biology techniques used in wastewater treatment: An overview

Identification of microorganisms by conventional methods requires the isolation of pure cultures followed by laborious characterization experiments. These procedures are therefore inadequate for study of the biodiversity of a natural or engineered ecosystem. A new set of molecular techniques developed during the 1990s revolutionized microbial ecology research. Among these techniques, cloning and the creation of a gene library, denaturant gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization with DNA probes (FISH) stand out. Cloning provides very precise taxonomical information, but is time consuming and requires specialized personnel and so its introduction in wastewater treatment has been slow. DGGE is a rapid and simple method that provides characteristic band patterns for different samples, allowing quick sample profiling, while retaining the possibility of a more thorough genetic analysis by sequencing of particular bands. FISH makes possible to identify microorganisms at any desired taxonomical level, depending on the specificity of the probe used. It is the only quantitative molecular biology technique, although quantification is either complex or tedious and subjective. Combination with a confocal laser-scanning microscope allows the visualization of three-dimensional microbial structures (granules, biofilms). The methods discussed have deepened our understanding of the microbiology of biological wastewater treatment. PCR-based methods (cloning and DGGE) have proved suitable for identifying the microorganisms that form the sludge. Both DGGE and FISH have been extensively employed. FISH is currently being used for elucidation of the composition, quantification and distribution of different bacterial groups in granules and biofilms, as well as their structure and architecture.     

probeBase: an online resource for rRNA-targeted oligonucleotide probes

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.     

Detection of activity among uncultured Actinobacteria in a drinking water reservoir

The abundance, identity and activity of uncultured Bacteria and Actinobacteria present in a drinking water reservoir (North Pine Dam, Brisbane, Australia) were determined using a combination of fluorescence in situ hybridization (FISH) alone or with catalysed reporter deposition (CARD-FISH) with microautoradiography. The CARD-FISH technique was modified relative to previous described procedures and performed directly on gelatine cover slips in order to allow simultaneous combination with microautoradiography. Almost twofold higher numbers of microorganisms could be identified as either Bacteria or Actinobacteria using the CARD-FISH technique as compared with the traditional FISH technique. A combination of FISH or CARD-FISH with microautoradiography showed generally higher activity among the Actinobacteria than among all Bacteria. Another important observation was that many cells within the FISH-negative populations of both Actinobacteria and Bacteria were actively assimilating thymidine. Thus, great care should be taken when extrapolating the active fraction of a prokaryotic community to be equivalent to the FISH-detectable population in such environments. Bacterial groups within Actinobacteria produce the odours geosmin and 2-methylisoborneol, which lower the quality of surface water when used for drinking. The results indicate that combined microautoradiography and CARD-FISH may serve as an effective tool when studying identity and activity of microorganisms within freshwater environments.     

FISH技术在微生物生态学中的研究及进展

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。