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实时荧光定量PCR的应用和进展 总被引:7,自引:0,他引:7
实时荧光定量PCR技术通过检测PCR产物中荧光讯号强度来达到定量的目的,该技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高等特点,目前已在动植物基因工程,微生物和医学领域中得到广泛应用。本文对实时荧光定量PCR技术的原理、优缺点及近年来新兴起的荧光探针的原理、优缺点进行了评述,重点及创新点是对实时荧光定量PCR技术在动植物基因工程,微生物和医学领域的应用进行了比较全面的综述,并对实时荧光定量PCR技术的普及应用及在基因诊断领域的前景做了进一步的展望。 相似文献
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实时荧光定量PCR技术及其在昆虫学研究中的应用 总被引:1,自引:0,他引:1
实时荧光定量PCR(real-time fluorescent quantitative polymerase chain reaction, FQ-PCR)是一种利用荧光信号实时监测体外DNA分子PCR复制过程中每个循环的扩增产物,从而实现对DNA模板进行定量分析的技术,具有准确、快速、灵敏、特异等优点,在动植物基因工程、动植物检疫、微生物鉴定与分类、食品安全检测和医学等领域中得到广泛应用。本文对实时荧光定量PCR技术的原理、优缺点及近年来新的荧光探针的原理、类型进行了评述,并对实时荧光定量PCR技术在昆虫学研究中的应用进行了综述。 相似文献
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FISH技术在微生物生态学中的研究及进展 总被引:3,自引:0,他引:3
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。 相似文献
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实时荧光定量PCR技术在鱼类病害研究中的应用 总被引:1,自引:0,他引:1
实时荧光定量PCR技术是一种新的核酸定量技术,通过检测PCR产物中荧光信号强度达到定量的目的,与常规PCR相比,具有无污染、特异性强、检测灵敏、定量准确等特点,该技术在分子诊断、动植物检疫等方面得到了广泛的应用.目前水产养殖业处于飞速发展时期,其中鱼类的病害问题也日益突出,为了预防和控制鱼类病害,实时荧光定量PCR技术已逐渐应用于鱼类病害的研究中.该文将从实时荧光定量PCR的技术原理、主要类型以及实时荧光定量PCR技术在鱼类病害研究的应用研究作一综述. 相似文献
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实时荧光定量PCR技术被广泛应用于实验研究、临床检测中。与普通的PCR相比,实时荧光定量PCR技术具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点。我们综述了实时荧光定量PCR技术的原理、定量方法,及其在传染性疾病检测研究中的应用。 相似文献
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3 次连续重复提取DNA 能较好反映土壤微生物丰度 总被引:7,自引:1,他引:6
【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。 相似文献
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Cassler M Peterson CL Ledger A Pomponi SA Wright AE Winegar R McCarthy PJ Lopez JV 《Microbial ecology》2008,55(3):384-394
In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms. 相似文献
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Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. 相似文献
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We show that inferring the taxa-abundance distribution of a microbial community from small environmental samples alone is
difficult. The difficulty stems from the disparity in scale between the number of genetic sequences that can be characterized
and the number of individuals in communities that microbial ecologists aspire to describe. One solution is to calibrate and
validate a mathematical model of microbial community assembly using the small samples and use the model to extrapolate to
the taxa-abundance distribution for the population that is deemed to constitute a community. We demonstrate this approach
by using a simple neutral community assembly model in which random immigrations, births, and deaths determine the relative
abundance of taxa in a community. In doing so, we further develop a neutral theory to produce a taxa-abundance distribution
for large communities that are typical of microbial communities. In addition, we highlight that the sampling uncertainties
conspire to make the immigration rate calibrated on the basis of small samples very much higher than the true immigration
rate. This scale dependence of model parameters is not unique to neutral theories; it is a generic problem in ecology that
is particularly acute in microbial ecology. We argue that to overcome this, so that microbial ecologists can characterize
large microbial communities from small samples, mathematical models that encapsulate sampling effects are required. 相似文献
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Combinations of microscopy and molecular techniques to detect, identify and characterize microorganisms in environmental and medical samples are widely used in microbial ecology and biofilm research. The scope of these methods, which include fluorescence in situ hybridization (FISH) with rRNA-targeted probes, is extended by digital image analysis routines that extract from micrographs important quantitative data. Here we introduce daime (digital image analysis in microbial ecology), a new computer program integrating 2-D and 3-D image analysis and visualization functionality, which has previously not been available in a single open-source software package. For example, daime automatically finds 2-D and 3-D objects in images and confocal image stacks, and offers special functions for quantifying microbial populations and evaluating new FISH probes. A novel feature is the quantification of spatial localization patterns of microorganisms in complex samples like biofilms. In combination with '3D-FISH', which preserves the 3-D structure of samples, this stereological technique was applied in a proof of principle experiment on activated sludge and provided quantitative evidence that functionally linked ammonia and nitrite oxidizers cluster together in their habitat. This image analysis method complements recent molecular techniques for analysing structure-function relationships in microbial communities and will help to characterize symbiotic interactions among microorganisms. 相似文献
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Species-specific enumeration of mixed community is invaluable as it facilitates a better understanding of the significance of the individual strains, their interactions, and the underlying mechanisms of community dynamics. Mixed microbial community has been characterized by microbiological, biochemical, or molecular biology-based methods. While microbiological and biochemical techniques do not provide adequate quantitative information of the members of the consortia and require additional techniques for a more comprehensive analysis, molecular biology-based methods analyze the microbial consortium based on specific DNA sequences and do not require isolation and culturing of bacteria for quantitative analysis. These methods outshine conventional culture-based techniques in terms of better sensitivity, reproducibility, and reliability. Quantitative molecular biology methods have been classified as PCR-based and probe hybridization methods. The PCR-based methods includes quantitative real-time PCR and terminal restriction fragment length polymorphism, while fluorescent in situ hybridization and DNA microarrays fall under probe hybridization methods. The workflow, the quantification methods, and their potential applications are discussed in this review by highlighting their advantages and possible limitations. 相似文献
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A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17alpha-estradiol, 17beta-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats. 相似文献
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定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率。qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高速等密度梯度离心将被同位素标记的重链核酸与未被标记的轻链核酸进行分离,并对所有组分微生物类群进行绝对定量和测序分析,基于GC含量和未标记处理DNA密度曲线量化参与吸收转化的DNA同位素丰度。本文重点阐述qSIP的技术原理、数据分析流程及其在微生物生态学研究中的应用进展,并对该技术存在的问题进行了分析和展望。 相似文献