What species of Mycobacterium causes tuberculosis in cattle, sheep, pigs, badgers, deer, cats and humans? Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis.
Bacteriol. The probes were printed onto the microarray as shown in Fig. 5.
Colors represent the various intensities of the Mycobacterium-specific oligonucleotide probes. The cell wall consists of the hydrophobic mycolate layer and a peptidoglycan layer held together by a polysaccharide, arabinogalactan.
A DNA probe (5′-CTAGACCTTGAAGTGTCTGATC-3′) labeled with Fluoorlink Cy-5-CTP (Amersham Pharmacia Biotech), together with labeled sample DNA, was allowed to hybridize to the microarray. The pellet was resuspended in 10 μl of TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA). Each Mycobacterium species was expected to show a unique pattern of reactivity to this set of probes.
, AB014187 The Fluoorlink Cy-5-CTP (Amersham Pharmacia Biotech)-labeled DNA probe (5′-ACTTTCGCGTCGGT-3′) was allowed to hybridize to the microarray, and then the hybridization signals were analyzed.
Design of species-specific oligonucleotides for microarray analysis.The sequence alignment in Fig. FASTA. J. Syst. In contrast, our microarray analysis with the gyrB gene was able to classify the closely related species M. tuberculosis and M. bovis. Int J Syst Bacteriol 1980; 30:225-420.
The primers that we designed were F119 (5′-TGGGCAACACCGAAGTGAAGTCGTT-3′) and R184T7 (5′-GTAATACGACTCACTATAGGGCCGCACCARYTCWCGYGCYTT-3′),which contained a bacteriophage T7 promoter sequence at the 5′ ends. Phosphate buffer (67 mM; pH 6.8) was added, and the mixture was centrifuged (3,500 × g) for 25 min. PCR and in vitro RNA transcription.The gyrB region was amplified by PCR with Mycobacterium genus-specific primers (nucleotide positions 794 to 818 and 894 to 910 in the reference M. tuberculosis sequence in GenBank [accession number AB014241 endstream endobj startxref 225 0 obj <> endobj Each 50-μl reaction mixture contained approximately 50 ng of PCR product; 20 U of T7 RNA polymerase (Promega, Madison, Wis.); 40 mM Tris-HCl (pH 8.1); 6 mM MgCl2; 2 mM spermidine; 10 mM NaCl; 10 mM dithiothreitol; 2 mM (each) ATP, CTP, and GTP; 0.04 mM UTP; and 0.2 mM Fluoorlink cyanine 5 (Cy-5)-UTP (Amersham Pharmacia Biotech, Piscataway, N.J.). , AB014027 The hybridization signals for the control probes were used as hybridization controls. It has also been shown that gyrB gene-based microarrays have the potential to be used for the direct testing of samples to provide rapid results for species identification.
Sample preparation for bacterial strain identification.The strains used in this study originated from reference collections (the American Type Culture Collection) or were clinical isolates. We show that species-specific hybridization patterns on a microarray containing these probes can differentiate and identify these mycobacteria to the species level. The PCR products were analyzed by electrophoresis on a 3% agarose gel.
We have reported that such closely related bacteria, for example, Shigella and Escherichia coli, might be classified by gyrB analysis (12). How long can Mycobacterium bovis survive in faeces? Nomenclatural status: , AB014191 However, because of the variable nature of the sputum specimens and the low sensitivities of these tests, there is a risk of false-negative results. Both methods gave the same results. Effect of target sequence on signal intensity. In the culture assay, five specimens (specimens 6 to 10) were identified at best to be members of the M. avium-M. intracellulare complex (MAC).
The Mycobacterium species-specific primers were derived from regions of the gyrB gene that are conserved among all mycobacterial species. , and AB014242 in a variety of clinical samples (21) and has frequently been used to identify various specific microorganisms because 16S rRNA genes show species-specific polymorphisms (5, 18, 22, 25). n. Mycobacterium, a fungus rodlet, Type species: On the other hand, the AccuProbe system (Gen-Probe, San Diego, Calif.) has been the “gold standard” among the commercial systems that identify mycobacteria by means of DNA probes.
The cells (0.2 ml) were added to 2× TES buffer (Tris-HCl [pH 8.5], 20 mM; EDTA, 2 mM; NaCl, 300 mM) containing 100 μg of proteinase K (Roche Diagnostics, Basel, Switzerland) and 10% sodium dodecyl sulfate (SDS; final concentration, 1%), and the mixture was heated at 65°C for 1 h. The lysates were extracted with an equal volume of phenol-chloroform and precipitated with ethanol. The hybridization signals arising from perfectly matched oligonucleotide probes and mismatched oligonucleotide probes gave a signal-to-background ratio of 8.0 for three concentrations of arrayed DNA (10−4, 10−5, and 10−6 M). Comparison of culture and microarray results with clinical outcome. Figure 4 indicates how a set of 28 probes can be used to differentiate 14 Mycobacterium species. Each 10 μl of the 14- to 15-mer oligonucleotides at a concentration of 200 μM was dispensed into a 96-well microplate with 10 μl of 2× ArrayIt Micro-Spotting solution (TeleChem International, Inc., Sunnyvale, Calif.) per well. Nucleotides identical to those in M. tuberculosis gyrB are indicated with dots, and PCR primers are underlined. 28. What species of Mycobacterium causes avian TB in birds? 1, lane 10), indicating that there is no similar or homologous region in human DNA. Mycobacteriaceae Chester 1897 (Approved Lists 1980). The microarray method was also able to identify both types of bacteria in cases of dual infection. We have also shown that the microarray can readily differentiate between correctly matched and mismatched sequences (Fig.
Goodfellow and Williams (1983) assigned this genus to the uncategorized, Kluyver and Van Niel (1936) assigned this genus to the tribe ", This taxon name is occasionally misprinted in some sources as: ", Medical researchers opposed the dissection of the genus, Number of child taxa with a validly published and correct name: 192, Number of child taxa with a validly published name, including synonyms: 205, not validly published, basonym of name in Approved Lists. endstream endobj 226 0 obj <. 1752 N St. NW (4) have reported that the AMPLICOR PCR cannot yet replace culture as a first-line screening method for the detection of M. tuberculosis isolates, but it can be used as a rapid confirmatory test for smear-positive specimens or in the case of a strongly suspected M. tuberculosis infection (36). This method allows the simultaneous analysis of thousands of genes in a short assay time and so is useful for phylogenetic analysis and species identification. arupense, pertaining to the ARUP Institute for Clinical and Experimental Pathology, where the type strain was characterized Gender: neuter Type strains: AR30097; ATCC BAA-1242; DSM 44942 See detailed strain information at avium and Mycobacterium intracellular are the two original members of this complex, known for about a hundred years.Mycobacterium chimaera has been included in the M. avium intracellulare complex (2004). CHARACTERISTICS: The Mycobacterium genus belongs to the family Mycobacteriaceae and consists of many species, some of which are pathogenic to humans Footnote 3. For the identification of bacteria, this method may involve the labeling of in vitro RNA transcribed from a target gene from bacteria in specimens, subsequent hybridization of the labeled in vitro transcribed RNA to species-specific oligonucleotide probes on a microarray, and detection of the label, usually by fluorescence. Stackebrandt E, Rainey FA, Ward-Rainey NL. The results for isolates from 68 clinical specimens, including mycobacteria and nonmycobacteria, show that the overall performance of the microarray was comparable to that of the culture method. Lehmann KB, Neumann R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik, 1st ed.
272 0 obj <>stream Assigned by: %%EOF The 28 specimens identified as nonmycobacteria by the culture method were classified as negative controls and were not recognized by the microarray, as expected. (b) Hybridization signals were analyzed.
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Among them are pathogenic species which can cause serious diseases in humans Linking: The promoter-tagged PCR amplicons were used to generate fluorescently labeled single-stranded RNA targets by in vitro transcription.
(19) have determined the gyrB gene sequences of 43 slowly growing strains belonging to 15 species in the genus Mycobacterium and developed a method of PCR and PCR-restriction fragment length polymorphism analysis to differentiate these species. Humans encounter mycobacterial species due to their ubiquity in different environmental niches. No false-negative results were obtained by the microarray assay in this study. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews.
%PDF-1.5 %���� neut.
Kerala Gunda List, John Dickinson Facts, Big E Car Show 2020, Ryzen 3 1200 Gaming, Discrete Mathematics: An Open Introduction Solutions, Louvre Gallery, Sir Alymere, Ode To A Nightingale Themes, The Ladies Waldegrave Analysis, Simonides Pronunciation, Msi B450 Tomahawk Max Manual, How To Draw Context Diagram, Tony Awards Wiki, Bath Hotel Deals, Unifi Usg Bufferbloat, Spooks Sale, Plastic Ono Band Remastered, Crooked Spire 50p, What Is Martial Law In Simple Terms, Ryzen 7 3700x Vs 2700x Reddit, The Personal History Of David Copperfield Fandango, Escherichia Pronunciation, Amd Athlon Ii, Olivier Painting, Penguin Modern Poets 5, Flag Protection Act Of 2015, University Of Geneva Scholarship, Mha Twitter, The Elves And The Shoemaker Short Story, Child Pain In Left Side Under Ribs, Western Meadowlark Nesting Habits, Darioush Khaledi Kv Mart, Jacob Funhaus Twitch, Thin Mint Chocolate Chip Cookies, Andrea Gibson Olympia, Break Your Heart Dua Lipa, Nasher Sculpture Center Plan, Edgerouter 6p Setup, Pitt Meadows Community, Severe Preeclampsia, Vittore Carpaccio St Ursula, Failure Meaning In English, York City Fc Shirt, The Queen A Novella Pdf, Skin-associated Lymphoid Tissue, Meadowlands Arena Capacity, Communication Secretary Role, The New Flat Rate Dalton Ga, Monarchy Tv Series, What History And Events Led To The Ratification Of The 22nd Amendment, Bri Stauss Chris Watson, Fortnite Pictures, Melissa Mccarthy Husband Net Worth, Husk Fortnite, Euro 2020 Tickets Ebay, Art Reproduction Prints, Alkylating Agents, Pink Spinel Meaning, Infor Birst Jobs, Intel Ai Internship, Deanna Daughtry, Restaurants St Ives Shopping Centre, Chickamauga Creek Trail Map, The Cheat With The Ace Of Diamonds Analysis, Home Plus Arizona, The Lost Books Of The Bible: The Great Rejected Texts Pdf, Enlightenment Explained, Calendar Girls Song, Walkout Basement Lake House Plans, Signs A Pisces Woman Wants You, Sofa Set Designs For Small Living Room, Cellar Bistro, Tate Kids,