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Query: UNIPROT:P06889 (Mol)
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As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.
Mol Cell Probes 1993 Jun
PMID:Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format. 839

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.
Mol Cell Probes 1993 Jun
PMID:PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells. 839 1

Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.
Mol Cell Probes 1993 Feb
PMID:Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA. 845 41

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic 'tool-box' for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.
Mol Microbiol 1993 Feb
PMID:DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. 845 66

Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. We show here that the 183-amino-acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix DNA-binding motif--properties associated with repressors of temperate phages. We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria. Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic-resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines.
Mol Microbiol 1993 Feb
PMID:Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria. 845 67

An express method has been elaborated for identification of human and bovine species mycobacteria in the polymerase chain reaction. The high specificity of the technique and simplicity of material preparation for research make the experimental procedure simple and quick, permitting one to identify Mycobacterium tuberculosis and Mycobacterium bovis directly in pathogenic material.
Mol Gen Mikrobiol Virusol
PMID:[Development of a method for identifying bovine and human types of mycobacteria using the polymerase chain reaction]. 851 47

A multiplex polymerase chain reaction has been developed which is able to distinguish Mycobacterium tuberculosis from other members of the M. tuberculosis complex. The assay is based on the simultaneous amplification of two different targets: a 396bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 245bp fragment from the M. tuberculosis complex insertion sequence IS986. Results have been obtained for 54 mycobacterial strains including five non-M. tuberculosis complex isolates. All 49 strains of the M. tuberculosis complex were positive for IS986 but only the 27 M. tuberculosis isolates were positive for both IS986 and mtp40.
Mol Cell Probes 1995 Oct
PMID:A multiplex polymerase chain reaction for distinguishing Mycobacterium tuberculosis from Mycobacterium tuberculosis complex. 856 67

A nested polymerase chain reaction (PCR) procedure was devised for identification of mycobacteria. The outer reaction exploiting genus-specific sequences on the 16S rRNA gene was able to amplify specifically strains of the genus Mycobacterium. The identification of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare was accomplished by selective reamplification of the outer PCR product in three distinct inner amplifications exploiting species-specific primers mapping to a hypervariable region of mycobacterial 16S rRNA. Detection of mycobacteria, other than those for which species-specific primers were used, was accomplished by adding a supplementary genus-specific upper primer to one of the inner reactions. Specificity of amplification was confirmed for clinical isolates and reference strains of different mycobacterial species with the exception of a M. intracellulare type 7 strain which was recognized as M. avium. The amplification protocol presented thus provides a reliable and cost-effective way for identification of clinically relevant mycobacteria.
Mol Cell Probes 1995 Oct
PMID:Identification of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare by selective nested polymerase chain reaction. 856 72

The systems participating in detoxification of reactive oxygen intermediates in Mycobacterium tuberculosis are believed to play a dual role in the biology of this highly adapted human pathogen: (i) they may contribute to the survival of this bacterium in the host; and (ii) alterations in the gene encoding catalase/peroxidase have been linked to this organism's resistance to the front-line antituberculosis drug isoniazid. These relationships prompted us to extend investigations of the oxidative-stress-response systems in M. tuberculosis by analysing the alkyl hydroperoxide reductase gene ahpC and its putative regulator oxyR. Surprisingly, the oxyR gene was found to be inactivated by multiple lesions in M. tuberculosis H37Rv. These alterations were observed in all M. tuberculosis strains tested, and in members of the M. tuberculosis complex: Mycobacterium bovis BCG, Mycobacterium africanum, and Mycobacterium microti. The corresponding region carrying these genes in Mycobacterium leprae, an organism not sensitive to isoniazid, has a complete oxyR gene divergently transcribed from ahpC. An increase in minimal inhibitory concentration for isoniazid was observed upon transformation of M. tuberculosis H37Rv with cosmids carrying the oxyR-ahpC region of M. leprae. In keeping with the observed inactivation of oxyR, transcriptional activity of the corresponding region in M. tuberculosis was an order of magnitude lower than that of the oxyR gene from M. leprae. While the loss of this putative regulator of oxidative-stress response in M. tuberculosis is paradoxical considering the fact that survival in host macrophages is regarded as a critical feature of this pathogen, it offers a partial explanation for the exquisite sensitivity of M. tuberculosis to isoniazid.
Mol Microbiol 1995 Sep
PMID:Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene: implications for sensitivity to isoniazid. 859 38

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.
Mol Microbiol 1995 Sep
PMID:Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. 859 39


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