Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis.

1991 ◽  
Vol 29 (10) ◽  
pp. 2163-2168 ◽  
Author(s):  
P Del Portillo ◽  
L A Murillo ◽  
M E Patarroyo
2018 ◽  
Vol 475 (21) ◽  
pp. 3493-3509 ◽  
Author(s):  
Dhakaram Pangeni Sharma ◽  
Ramachandran Vijayan ◽  
Syed Arif Abdul Rehman ◽  
Samudrala Gourinath

The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.


2000 ◽  
Vol 38 (8) ◽  
pp. 2962-2965 ◽  
Author(s):  
Paul W. Whitby ◽  
Karen B. Carter ◽  
Kenneth L. Hatter ◽  
John J. LiPuma ◽  
Terrence L. Stull

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans andB. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.


1996 ◽  
Vol 40 (8) ◽  
pp. 1790-1795 ◽  
Author(s):  
G A Cangelosi ◽  
W H Brabant ◽  
T B Britschgi ◽  
C K Wallis

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well.


2017 ◽  
Vol 45 (18) ◽  
pp. 10884-10894 ◽  
Author(s):  
Kailu Yang ◽  
Jeng-Yih Chang ◽  
Zhicheng Cui ◽  
Xiaojun Li ◽  
Ran Meng ◽  
...  

2006 ◽  
Vol 188 (12) ◽  
pp. 4271-4287 ◽  
Author(s):  
Richard C. Huard ◽  
Michel Fabre ◽  
Petra de Haas ◽  
Luiz Claudio Oliveira Lazzarini ◽  
Dick van Soolingen ◽  
...  

ABSTRACT In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, long-sequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n = 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for “Mycobacterium canettii,” Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC.


2000 ◽  
Vol 38 (2) ◽  
pp. 737-744 ◽  
Author(s):  
Luis Miguel González ◽  
Estrella Montero ◽  
Leslie J. S. Harrison ◽  
R. Michael E. Parkhouse ◽  
Teresa Garate

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1,272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to bothT. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.


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