The 16Sribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction

Cox, Robert A.; Kempsell, Karen E.; Fairclough, Lynne; Colston, M. Joseph

doi:10.1099/00222615-35-5-284

Cited by 37 publications

(40 citation statements)

References 14 publications

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“…Members of the M . tuberculosis complex are also very closely related (98% similarity in 16s rRNA sequences) to other slow-growing mycobacteria (Rogall et al, 1990;Stahl & Urbance, 1990;Cox et al, 1991). This high degree of similarity in sequence suggests that the principal features of the leader region of the M .…”

Section: Discussionmentioning

confidence: 83%

“…All chemicals and enzymes were obtained from suppliers described previously (Cox et al, 1991). T7 sequencing kit was supplied by Pharmacia.…”

Section: Methodsmentioning

confidence: 99%

“…Cfoned M. leprae cDNA. Clones of the M. leprae rRNA operon were isolated from a cosmid Lawrist 4 library (Cox et al, 1991). The putative leader sequence of precursor rRNA and part of the coding sequence was found in a 901 bp Sau3AIPstI fragment, which was sequenced by standard methods.…”

Section: Methodsmentioning

confidence: 99%

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The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

Kempsell¹,

Estrada

et al. 1992

Journal of General Microbiology

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Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16s rRNA coding region and continuing to the start of the 23s rRNA coding region. The 16s rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia culi. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase I11 processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 1 6 s rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. Zeprae; although the principal features are common to both species there are notable differences.

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Section: Discussionmentioning

confidence: 83%

“…All chemicals and enzymes were obtained from suppliers described previously (Cox et al, 1991). T7 sequencing kit was supplied by Pharmacia.…”

Section: Methodsmentioning

confidence: 99%

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The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

Kempsell¹,

Estrada

et al. 1992

Journal of General Microbiology

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“…Sequence analyses of bacterial 16S rRNA genes were performed as previously described (13,14). Briefly, bacterial DNA was extracted from samples, and sequencing templates were prepared by PCR with one of the following primer sets: (i) C70f and B37r, (ii) H35f and H587r, (iii) C70f and H676r, (iv) H276f and cg4r, and (v) H276f and p13Br (3,13,33,34). Templates were purified on 3.5% polyacrylamide gels, and the gene sequence was determined by using either the Taq Dye Deoxy Termantory Cycle Sequencing Kit (Applied BioSystems, Inc., Foster City, Calif) or the TAQuence Cycle Seq Kit (U.S. Biochemicals, Cleveland, Ohio) (10,16).…”

Section: Bacterial Isolatesmentioning

confidence: 99%

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species

et al. 2001

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Over the past decade, several Helicobacter species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents. In this report, we describe a novel urease-negative helicobacter, Helicobacter typhlonius sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections. H. typhlonius is genetically most closely related to H. hepaticus. Like H. hepaticus, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H. hepaticus. Notably, H. typhlonius does not produce urease or H 2 S nor does it hydrolize indoxyl-acetate. Compared to other Helicobacter species that commonly colonize rodents, H. typhlonius was found to be less prevalent than H. hepaticus and H. rodentium but as prevalent as H. bilis. H. typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

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“…The specificity of the assay for detection of DDS susceptibility was determined by comparing the HD-DDS-ML profiles generated from M. leprae strains to the DNA sequence data from the SRDR of folP1 and the MFP DDS susceptibility data from these strains. Bacterial extracts testing negative by HD-DDS-ML analysis were tested by PCR for 16S ribosomal DNA common to all bacteria to rule out the presence of general PCR inhibitory activity (3).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous Detection of Mycobacterium leprae and Its Susceptibility to Dapsone Using DNA Heteroduplex Analysis

et al. 2001

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Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the folP1 gene of Mycobacterium leprae, and definitive evidence linking these mutations with DDS resistance in M. leprae has been obtained. Based on these mutations, a heteroduplex DDS M. leprae (HD-DDS-ML) assay was developed for the simultaneous detection of M. leprae and of its susceptibility to DDS. The assay relies on the PCR amplification of an M. leprae-specific 231-bp fragment of folP1 containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis. M. leprae was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from M. leprae-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for M. leprae in a comparison with results obtained from 14 species of mycobacteria other than M. leprae and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100 M. leprae organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of M. leprae and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.Prior to the development and implementation of multidrug therapy (MDT) for leprosy, most patients were treated with dapsone (DDS) monotherapy. During this period, DDS-resistant strains of Mycobacterium leprae were identified and DDSresistant leprosy became a significant problem for leprosy control programs (10, 12, 16). The currently recommended leprosy treatment with DDS, clofazimine, and rifampin (11) should control the spread of drug-resistant strains; however, DDSresistant strains of M. leprae continue to be reported even in areas of the world with successful implementation of MDT (1, 5, 13).M. leprae has yet to be cultivated on artificial medium; therefore, to obtain drug susceptibility patterns, the bacteria must be tested in Shepard's mouse footpad (MFP) assay (19). Because of the cumbersome nature of this drug-screening method, comprehensive estimates of drug resistance in leprosy have been difficult to obtain. Rec...

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The 16Sribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction

Cited by 37 publications

References 14 publications

The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae prefnsor rRNA

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species

Simultaneous Detection of Mycobacterium leprae and Its Susceptibility to Dapsone Using DNA Heteroduplex Analysis

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