【摘要】 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更多还原

【Abstract】 During recent years,molecular techniques such as PCR and denaturing gradient gel electrophoresis (DGGE) or DNA sequencing have revolutionized all fields of microbiology,and sensitive detection and exact identification of bacteria are possible.Fluorescence in situ hybridization (FISH) using 16SrRNA probes does not rely on PCR amplification,and as such provides a useful complementary technique to DGGE for the analysis of organisms. Because of FISH allowing nucleic acid sequences to be examined inside cells without altering the cell’s morphology or the integrity of its various compartments and providing information about number,spatial distribution and cellular environment,it has become a powerful tool for phylogenetic,ecologic,diagnostic and environmental studies in microbiology. Fluorescence in situ hybridization can detect nucleic acid sequences by a fluorescently labeled probe that hybridizes specifically to its complementary target sequence within the intact cell. Its general procedure in FISH analysis of microorganisms is as follows: (1) fixation of the specimen; (2) preparation of the sample,possibly including specific pretreatment steps; (3) hybridization with the respective probes for detecting the respective target sequences; (4) washing steps to remove unbound probes; (5) mounting,visualization and documentation of results. Because FISH gives a detailed picture of the microenviroments without any selective purification or amplification steps, there is a large scope of FISH applications. It has been extensively used in the field of environmental microorganisms diversity such as the investigation of microbial communities of aquatic habitats and soil habitats. Most obligate microbial symbionts are as-yet uncultured. Using the 16SrRNA approach,they can be identified and phylogenetically classified.Using different FISH strategies,bacterial endosymbionts were detected in many microorganisms. Population analysis by FISH has proven particularly useful for description of the normal flora or that of mixed microbial infections. This has been shown for medicine research such as oral cavity,gastro-intestinal flora,respiratory tract infections. It should be pointed out that FISH is not free of biases as with other molecular methods.The most striking problem is autofluorescence of microorganisms themselves.In addition,accuracy and reliability of FISH is highly dependent on the specificity of the oligonucleotide probe. Fish provides information about the presence,number,morphology and spatial distribution of microorganisms.We believe that FISH as a powerful tool will make more contributions to the assessment of the population structure of complex microbal communities.更多还原