• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.333-339
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• 2011

Loop-mediated isothermal amplification (LAMP) was developed to detect Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacterium (NTM) genomic DNA in blood samples of Korea native cattle. A set of four primers, two outer and two inner, were designed from M. bovis and M. avium genomic DNA targeting the IS6110 and 16S rRNA gene, respectively. Based on 85 Intradermal Tuberculin Test (ITT) positive blood sample and using conventional PCR and LAMP, the agreement quotient (kappa), which measures agreement beyond chance were 0.93 (conventional PCR) and 0.97 (LAMP), respectively. The detection limit of the LAMP method was $2.0{\times}10^2$ copy/ml M. bovis and M. avium cells, compared to $2.0{\times}10^3$ copy/ml M. bovis and M. avium cells for conventional PCR. These results suggest that the LAMP is a powerful tool for rapid, sensitive, and practical detection of MTC and NTM in blood samples of Korea native cattle.

• The Korean Journal of Cytopathology
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• v.12 no.1
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• pp.25-30
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• 2001

Tuberculous lymphadenitis is not uncommon in Korea. Therefore, an inexpensive, safe and rapid method is needed to diagnose the tuberculous lymphadenitis. Flne needle aspiration cytology Is a good method for this purpose, but has several limitations in the diagnosis of tuberculous lymphadenitis, especially when the presence of acid-fast bacilli is not proved. To evaluation the usefulness of the polymerase chain reaction with enzyme immunoassay technique in the detection of Mycobacterium tuberculosis (M. tuberculosis) In the cervical Iymph node asplrates, the authors performed fine needle aspiration cytology and M. tuberculosis PCR with enzyme immunoassay for mycobacterial DNA sequences from 15 cases of the fine needle aspirates. Cytomorphologically, the cases were categorized into three types: predominantly necrotic materials; typical epithelioid cell granulomas with or without slant cells and caseous necrosis; and non-tuberculous lesions, such as reactive lymphadenitis, abscess, metastatic carcinoma and malignant lymphoma. M. tuberculosis DNA was found in 8 of 15 cases by PCR with enzyme immunoassay. Negative findings on PCR were achieved in 7 cases, which revealed non-tuberculous tymphadenopathy. In conclusion, we suggest that M. tuberculosis PCR with enzyme immunoassay using the fine needle aspirates is a very useful tool for the diagnosis of tuberculous lymphadenitis.

• Tuberculosis and Respiratory Diseases
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• v.50 no.5
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• pp.599-606
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• 2001

Background : Since the advent of AIDS, tuberculosis has become a major public health problem in the western society. Therefore, it is essential that pulmonary tuberculosis be rapidly diagnosed. Light microscopic detection of acid-fast organisms in sputum has traditionally been used for rapidly diagnosing tuberculosis. However positive smears are only observed in about one-half to three-quarters of cases. Studies using PCR for diagnosing pulmonary tuberculosis disclosed several shortcomings suggesting an inability to distinguish between active and treated or inactive tuberculosis. In this study, the clinical significance of a PCR-based rapid technique for detecting Mycobacterium tuberculosis DNA in peripheral blood was investigated. Materials and Methods : From July 1, 1998 through to August 30, 1999, 59 patients with presumed tuberculosis, who had no previous history of anti-tuberculosis medication use within one year prior to this study were recruited and followed up for more than 3 months. AFB stain and culture in the sputum and/or pleural fluids and biopsies when needed were performed. Blood samples from each of the 59 patients were obtained in order to identify Mycobacterium Tuberculosis DNA by a PCR test. Results : 1) Forty five out of 59 patients had a final diagnosis of tuberculosis ; Twenty eight were confirmed as having active pulmonary tuberculosis by culture or biopsy. Four were clinically diagnosed with pulmonary tuberculosis. The other 13 patients were diagnosed as having tuberculous pleurisy (9) and extrapulmonary tuberculosis (4). 2) Fourteen patients showed a positive blood PCR test. The PCR assay correctly identified active tuberculosis in 13 out of 14 patients. The overall sensitivity and specificity of this blood peR assay for diagnosing tuberculosis were 29% and 93%, respectively. The positive predictive value was 93%, the negative predictive value was 29% and the diagnostic accuracy was 44%.3) Six out of 14(43%) patients with blood PCR positive tuberculosis were immunologically compromised hosts. 4) A simple chest radiograph in blood PCR positive tuberculosis patients showed variable and inconsistent findings. Conclusion : A peripheral blood PCR assay for Mycobacterium tuberculosis is not recommended as a screening method for diagnosing active tuberculosis. However, it was suggested that the blood PCR assay could contribute to an early diagnostic rate due to its high positive predictive value.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• Microbiology and Biotechnology Letters
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• v.30 no.2
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• pp.105-110
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• 2002

The sequence of 3,221 nucleotides immediately adjacent to rpsA gene encoding 30S ribosomal protein S1 of Brevibacterium ammoniagenes was determined. A putative open reading frame (ORF) of 2,670 nucleotides for a polypeptide of 889 amino acid residues and a TAG stop codon was found, which is located at a distance of 723 nucleotides upstream from rpsA gene with same translational direction. The deduced amino acid sequence of the ORF was found to be highly homologous to the DNA polymerase I of Streptomyces griseus (75.48％), Rhodococcus sp. ATCC 15963 (56.69％), Mycobacterium tuberculosis (55.46％) and Mycobacterium leprae (53.99％). It was suggested that the predicted product of the ORF is a DNA polymerase I with three functional domains. Two domains of 5 → 3 exonuclease and DNA polymerase are highly conserved with other DNA polymerase I, but 3 → 5 exonuclease domain is less conserved.

• Tuberculosis and Respiratory Diseases
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• v.81 no.3
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• pp.222-227
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• 2018

Background: Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea. Methods: Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence. Results: In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L. Conclusion: Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.

• 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.

• Journal of the Korean Chemical Society
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• v.52 no.3
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• pp.273-280
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• 2008

Mycobacterium tuberculosis (MTB) remains a major worldwide public health problem. In recent years, the incidence of MTB has been rising. Rapid and reliable diagnosis of Mycobacterium tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the MTB. In this research, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of MTB. Undiluted DNA (2.10 × 106 copy/mL), 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 (copy/mL) of MTB DNA were amplified by PCR and LAMP to determine the sensitivity of the assay. At results, the LAMP assay reported here has the advantages of rapid amplification, high sensitivity, and high specificity and will be useful for rapid and reliable clinical diagnosis of MTB in hospital clinical laboratory.

• The Journal of the Korean Society for Microbiology
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• v.34 no.5
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• pp.491-500
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• 1999

A recent study showed that comparative sequence analysis of rpoB DNAs could reveal natural relationships in genus Mycobacterium [J Clin Microbial. 37 (6). 1999]. rpoB DNAs showed interspecies variation and intraspecies conservation. Based on these data, we developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocols which enable species differentiation in genus Mycobacterium. When this assay was applied to 24 clinical isolates identified as M. avium complex (MAC) by biochemical test, these were successfully differentiated into M. avium and M. intracellulare. These results were concordant with those obtained by 16s rDNA analysis. It is the first report that PCR-SSCP analysis of rpoB DNA could be used for species differentiation of MAC strains.

• Tuberculosis and Respiratory Diseases
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• v.85 no.3
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• pp.256-263
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• 2022

Background: Mycobacterium tuberculosis (Mtb) is resistant to the β-lactam antibiotics due to a non-classical transpeptidase in the cell wall with β-lactamase activity. A recent study showed that meropenem combined with clavulanate, a β-lactamase inhibitor, was effective in multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). However, in Korea, clavulanate can only be used as drugs containing amoxicillin. In this study, we investigated the susceptibility and genetic mutations of drug-resistant Mtb isolates to amoxicillin-clavulanate and meropenem-clavulanate to improve the diagnosis and treatment of drug-resistant TB patients. Methods: The minimum inhibitory concentration (MIC) of amoxicillin-clavulanate and meropenem-clavulanate was examined by resazurin microtiter assay. We used 82 MDR and 40 XDR strains isolated in Korea and two reference laboratory strains. Mutations of drug targets blaC, blaI, ldtA, ldtB, dacB2, and crfA were analyzed by polymerase chain reaction and DNA sequencing. Results: The MIC₉₀ values of amoxicillin/clavulanate and meropenem/clavulanate in drug-resistant Mtb isolates were 64/2.5 and 16/2.5 mg/L, respectively. Gene mutations related to amoxicillin/clavulanate and meropenem/clavulanate resistance could not be identified, but T448G mutation was found in the blaC gene related to β-lactam antibiotics' high susceptibility. Conclusion: Our results provide clinical consideration of β-lactams in treating drug-resistant TB and potential molecular markers of amoxicillin-clavulanate and meropenem-clavulanate susceptibility.