• Korean Journal of Microbiology
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• v.48 no.4
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• pp.319-324
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• 2012

In this study, we investigated molecular divergences and phylogenetic characteristics of the 16S ribosomal RNA (rRNA) and RNA polymerase beta subunit (rpoB) gene sequences from the order Oscillatoriales (Cyanobacteria). The rpoB of Oscillatoriales showed higher genetic divergence when compared with those of 16S rRNA (p-distance: rpoB=0.270, 16S=0.109), and these differences were statistically significant (Student t-test, p<0.001). Phylogenetic trees of 16S rRNA and rpoB were generally compatible; however, rpoB tree clearly separated the compared Oscillatoriales taxa, with higher phylogenetic resolution. In addition, parsimony analyses showed that rpoB gene evolved 2.40-fold faster than 16S rRNA. These results suggest that the rpoB is a useful gene for the molecular phylogenetics and species discrimination in the order Oscillatoriales.

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.296-302
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• 2010

Microcystis (Cyanobacteria, Chroococcales) is one of the green tide-causing organisms in freshwaters, and some species produce microcystin that is hepatotoxin. In the aspects of freshwater quality controls and health concerns, therefore it is necessary to manage the harmful organisms. In the present study, RNA polymerase beta subunit (rpoB) gene sequences of Microcystis were determined and characterized in order to use a potential marker for the molecular detections of the species. Microcystis rpoB showed high divergences of DNA similarity and genetic distances when compared with those of 16S rRNA, and the molecular differences were statistically significant (Student t-test, p<0.05). Parsimony analyses showed the rpoB gene evolves more than 2-fold faster than 16S rRNA. In addition, phylogeny of the rpoB gene separated each M. aeruginosa strain more clearly compared with a 16S rRNA tree. This study found that the order Chroococcales, including Microcystis, has approximately two rRNA operons and single copy of the rpoB gene in their chromosomes. These results suggest that the rpoB gene is a useful marker for the molecular phylogenetics and the detection of Microcystis.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.268-274
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• 2011

Anabaena (Cyanobacteria, Nostocales) are important for water quality controls, because they are often responsible for freshwater green tides; moreover, some species are reported to produce hepatotoxin. In this study, we sequenced RNA polymerase beta subunit (rpoB) gene of Anabaena, and evaluated their sequences for the potential use of a molecular taxonomic marker in this taxon. Anabaena rpoB showed low DNA similarity and high genetic divergences when compared those of 16S rRNA, and the molecular differences were statistically significant (Student t-test, p<0.01). Parsimony analyses showed the rpoB gene evolves 4.8-fold faster than 16S rRNA. In addition, phylogeny of the rpoB gene separated each Anabaena strain more clearly compared with a 16S rRNA tree. These results suggest that the rpoB gene is a useful marker for the molecular phylogenetics and the species discrimination of Anabaena.

• Journal of the Korea Institute of Information and Communication Engineering
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• v.17 no.4
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• pp.1005-1009
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• 2013

Using DNA sequencing method, we analyzed mutations of rpoB (RNA polymerase beta subunit) rifampin-resistant Mycobaterium tuberculosis strains which were identified by conventional test at Masan National Hospital and The Korean Institute of Tuberculosis. Though it has been reported different mutations of rpoB region of rifampin-resistant M. tuberculosis strains in the south of Korea, it is not confirmed whether these mutations of rpoB region actually express rifampin resistance through experiment. We confirmed experimentally these mutations of rpoB region of M. tuberculosis strains induced rifampin-resistance through ampified rpoB by polymerase chain reaction (PCR) and cloning of mutant rpoB into rifampin sensitive-M. tuberculosis strain.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.853-869
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• 2000

Background : Following several decades of decline, the incidence of tuberculosis has recent1y begun to increase in many countries of this the control of this disease has been impeded by the emergence of multi-drug resistant tuberculosis (MDR-TB). The development of rapid diagnostic methods and effective new drugs are needed to control MDR-TB. One of the new drugs for MDR-TB is rifabutin (RBU) which has been known to be effective in some patients with MDR-TB. A few reports showed that some types of mutations of the rpoB gene, which were known to be present in 96-98% of rifampicin-resistant M. tuberculosis, were associated with the rifampicin-resistant but RBU-susceptible phenotype. This study was performed to investigate the correlation between RBU susceptibility and the patterns of rpoB gene mutations in Korean MDR-TB. Methods : Sixty-five clinical isolates of multi-drug resistant Mycobacterium tuberculosis, gathered from patients who visited the Asan Medical Center from July 1997 to June 1999, were investigated. Clinical responses to rifabutin-containing regimen were evaluated. An RBU susceptibility test and sequencing analysis of rpoB gene were performed, and the results were analyzed to confirm which mutations correlated with RBU-susceptible MDR-TB. Results : Fifty-three of 56 (95%) clinical isolates of MDR-TB had 60 mutations of the rpoB gene. The most frequent mutations were found at codon 531 (43%), and two mutations were combined in seven clinical isolates. Five of 53 (10%) clinical isolates showed the RBU-susceptible phenotype, and in them the characteristic patterns of point mutations were found at codon 509, 516, and 526. Conclusion : The frequency and pattern of mutations of the rpoB gene of Korean MDR-TB isolates were similar to those in western countries, where the prevalence of tuberculosis is low, but some show RBU-susceptible phenotypes. RBU-susceptible MDR-TB isolates showed the characteristic pattern of mutations of the rpoB gene which could be used to rapidly diagnose RBU susceptibility.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2012.10a
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• pp.991-993
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• 2012

We analyzed RNA polymerase beta subunit gene (rpoB) mutation of rifampin-resistant Mycobacteria through analysis of nucleotide sequence of rpoB DNA (351 bp) containing rifampin resistant region, $rif^r$. For this study, we collected rifampin-resistant Mycobacteria that were identified by conventional culture methods from Masan National Hospital and The Korean Institute of Tuberculosis. We performed sequencing of DNA nucleotides and analyzed rpoB gene of those rifampin-resistant Mycobacteria. From this analysis, we invcestigated diverse mutations of rpoB gene included rifampin-resistant gene, which were not reported, from those rifampin-resistant Mycobacteria.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.333-338
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• 2008

Computational analysis of partial rpoB gene sequence (777 bp) was done in this study to identify B. anthracis and its closely related species B. cereus and B. thuringiensis. Sequence data including 17 B. anthracis strains, 9 B. cereus strains, and 7 B. thuringiensis strains were obtained by searching databases. Those sequences were aligned and used for other computational analysis. B. anthracis strains were identificated by in silico restriction enzyme digestion. B. cereus and B. thuringiensis were not segregated by this method. Those sequencing and BLAST search were required to distinguish the two. In actual identification tests, B. anthracis strains could be identified by PCR-RFLP, and B. cereus and B. thuringiensis strains were distinguished by BLAST search with reliable e-value. In this study fast and accurate method for identifying three Bacillus species, and flow chart of identification were developed.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2012.05a
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• pp.913-915
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• 2012

RNA polymerase beta subunit gene (rpoB) mutation of rifampin-resistant Mycobacteria was analyzed using nucleotide sequence of rpoB DNA (351 bp) containing rifampin resistant region, $rif^r$. For this purpose, we collected rifampin-resistant Mycobacteria that were identified by conventional culture method from Masan National Hospital and The Korean Institute of Tuberculosis and performed analysis of nucleotide sequence of rpoB of them. We found various mutations of rpoB linked rifampin resistant gene from rifampin-resistant Mycobacteria. From this study, we identified mutations of different codons from codons that have been reported recently.

• Tuberculosis and Respiratory Diseases
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• v.50 no.3
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• pp.334-342
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• 2001

Background : RpoB gene mutations have been found in about 96-98% of rifampicin (RMP)-resistant Mycobacterium tuberculosis. Recent reports confirm that the in laboratory settings a rpoB gene mutation can be used as a surrogate marker for multi-drug resistant tuberculosis. However, its usefulness in clinical applications has not been evaluated. This study was performed to confirm whether mutation analysis of the rpoB gene of M. tuberculosis is useful in clinical settings. Methods : The medical records of 33 patients in whom rpoB gene analysis was conducted using an INNOLiPA Rif. TB assay (LiPA) from June, 1998, to July, 2000, at the Asan Medical Center were retrospectively reviewed in 33 patients. The clinical characteristics in addition to the drug susceptibility and LiPA results were analyzed. The drug susceptibility test was considered as a gold standard method for M. tuberculosis susceptibility and these results were compared with those of the rpoB gene study and sequencing analysis. Sequencing analysis of the rpoB gene was done in cases where there was a discrepancy between the results of the drug susceptibility an d rpoB gene study. Results : The mean age and sex ratio was $42{\pm}18$, and 24:9 (M:F), respectively. There were 19 RMP susceptible (58%) and 14 RMP-resistant cases (42%) according to the rpoB gene study. The mean time from the request to reporting the results of the rpoB gene study was $5.2{\pm}2.6$ days. The mean gap from reporting the rpoB gene study to reporting the susceptibility was $56{\pm}35$ days. Twenty-eight cases (85%) showed identical results compared with the drug susceptibility results, whereas five cases (15%) showed contradictory results. When compared with the sequencing analysis, of the five cases that showed contradictory results, two had LiP A analysis errors and the remaining three were identical to the sequencing results. The rpoB gene study was of assistance in choosing the appropriate drugs in 28 cases (85%). Conclusions : An rpoB gene study using an LiP A assay was useful in rapidly diagnosing RMP-resistant tuberculosis, which enabled a proper choice of the appropriate drugs in clinical practices. However, an LiPA assay always should be performed in conjunction with microscopy, culture, and susceptibility tests.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.842-851
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• 1996

Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.