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Detection of Mutations in Mycobacterium tuberculosis by a Dot Blot Hybridization Strategy

Genomic variation in any organism is of interest, because it may influence the phenotype of the organism. Special interest currently focuses on prokaryotic pathogens regarding mutations associated with resistance to therapeutic drugs, as well as those mutations involved in the evo ...

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Restriction Fragment Length Polymorphism Typing of Mycobacteria

In principle, restriction fragment length polymorphism (RFLP) typing can be applied to strains of all mycobacterial species for which suitable probes have been identified. International consensus has been achieved regarding the methodology of IS6110 RFLP typing of Mycobacter ...

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Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis

Culture filtrates obtained by in vitro cultivation of Mycobacterium tuberculosis have been studied for more than 20 years to identify and characterize proteins of immunological relevance. Culture filtrate preparations have been shown to induce a protective immune response in mi ...

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Analysis of the Capsule of Mycobacterium tuberculosis

Mycobacterium tuberculosis and the other pathogenic mycobacteria examined so far by electron microscopy are seen to be surrounded within host cells by an electron-transparent zone (ETZ). For Mycobacterium lepraemurium, this space between the phagosomal membrane of the infected ...

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Analysis of the Lipids of Mycobacterium tuberculosis

Mycobacterial cell wall ultrastructure has been studied through the use of negative staining, electron microscopy (1,2), freeze fracture (3), X-ray diffraction (4), differential scanning calorimetry (5,6), and electron spin resonance spectroscopy. Through the use of these techn ...

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In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis

Great progress has been made in the latter half of the twentieth century in the understanding of the immunology of tuberculosis and of strategies for chemotherapeutic management of this disease. Indeed, given the evidence that the dominant, and perhaps sole, ecologic niche of Mycobacteri ...

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Macrophage Virulence Assays

Mycobacterium tuberculosis is a facultative intracellular pathogen, which can survive and replicate within the host macrophage. It is transmitted via the aerosol route, which delivers the bacillus to the alveolus of the lungs. The initial phase of granuloma formation within the lung re ...

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Analysis of Mycobacterium-Infected Macrophages by Immunoelectron Microscopy and Cell Fractionation

The ability of pathogenic Mycobacterium to establish and maintain an infection in a host is dependent on their capacity to survive within phagocytes (1–3). Studies conducted on macrophage infections in culture have provided considerable insight into the mechanisms developed by the ...

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Real Time PCR Using Molecular Beacons: A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis

Molecular beacons are a novel family of hybridization probes, which emit fluorescence upon interaction with their target. They are hairpin-shaped oligonucleotides with a central part complementary to the target, flanked by two 5 6 base pair (bp) inverted repeats, which can form a stable ste ...

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Plasmid Vectors

Genetic manipulation of mycobacteria has historically been difficult. This is in large part due to the impenetrable nature of the cell wall, resulting in difficulty both in introducing DNA into the bacterium and subsequent isolation of intact plasmid DNA. In addition, the mycobacterial c ...

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Electronic Access Mycobacterium tuberculosis Sequence Data

The complete genome sequence of the well studied laboratory strain of Mycobacterium tuberculosis (H37Rv) has been published by the Sanger Centre (1), and this information will soon be complemented by the completion of a recent clinical isolate of M. tuberculosis (CDC1551/CSU93) by The Ins ...

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Proteomics

The term “proteomics” describes the technologies collectively used to define the protein complement of the genome or “proteome” (1,2). The recent growth of this discipline is reflected in the many review articles available (3–7). In addition to describing all the proteins encoded by the geno ...

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Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays

Completion of the sequence of the entire genome of strain H37Rv was a benchmark for Mycobacterium tuberculosis research (1). This achievement ushers in the era of genome-wide functional and comparative genomics for this organism. At present, the most powerful enabling technology of the p ...

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Storage of Mycobacterial Strains

The storage and maintenance of mycobacterial reference strains and clinical isolates are important parts of good laboratory practice in a mycobacterial laboratory. The storage of reference and control strains facilitates a reliable control on intra- and inter-test reproducib ...

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Isolation of DNA from Mycobacterium tubercolosis

Research into and identification of Mycobacterium tuberculosis can take on a number of facets, many of which involve the use of DNA at one stage or another. The quality and quantity of DNA required will depend on the end-use requirement. For example, good yields of pure, high-molecular-weight DNA un ...

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Safety in the Laboratory

The worldwide resurgence of tuberculosis (TB) has resulted in a rapid expansion in research efforts directed at a deeper understanding of the disease, the efficacy of vaccines, and improved drug targets. This has required the establishment of multiple new facilities to contain the pathog ...

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Extraction of RNA from Intracellular Mycobacterium tuberculosis: Methods, Considerations, and Applications

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term per ...

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Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages

Genetic analyses of pathogenic mycobacteria such as Mycobacterium tubeculosis and Mycobacterium bovis required improvement of existing methodologies for the generation of large representative libraries of mutants. Two basic methodologies have been used to generate mut ...

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Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB

Gene replacement and transposon mutagenesis are two complementary tools that have been widely used to perform genetic studies in various living organisms. In mycobacteria, and especially in the Mycobacterium tuberculosis complex, the lack of these tools has severely hampered the g ...

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Gene Replacement Mycobacterium tuberculosis and Mycobacterium bovis BCG

Much progress has been made in mycobacterial research in general and in mycobacterial genetics in particular during the past 10 yr. The complete genome sequences of two isolates of Mycobacterium tuberculosis, the widely distributed laboratory strain M. tuberculosis H37Rv (1) and a clin ...

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