• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2013년도 춘계학술대회
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• pp.1012-1014
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• 2013

DNA-DNA hybridization method with four oligonucleotide-specific probes was used simultaneously for differentiation and identification of four Mycobacterium species (Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii). This DNA-DNA hybridization method with 4 oligonucleotide-specific probes, which targets in the rpoB region of 4 Mycobacteria species, respectively, was tested on 322 clinical isolates. Using DNA-DNA hybridization method, we detected M. tuberculosis (282 strains), M. avim (7 strains), M. intracellulare (9 strains), and M. kansasii (3 strain) from 322 clinical isolates. This result was compared with conventional biochemical test and rpoB DNA sequence analysis of this clinical isolates. We confirmed identification of Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii with high sensitivity (100 %) and specificity (100 %). This DNA-DNA hybridization method could be performed within 4 hours at least. Therefore, we suggest that DNA- DNA hybridization method using 4 rpoB DNA probes of Mycobacteria could be used for accurate, rapid, convenient detection and identification of Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii in clinical samples.

• Tuberculosis and Respiratory Diseases
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• 제39권6호
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• pp.517-525
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• 1992

연구배경 : 1985년 Saiki등에 의해, 특정한 DNA를 연속적으로 복제할 수 있는 방법인 polymerase chain reaction (PCR)이 개발된 이래, PCR은 검체내에 극미량으로 존재하고 있는 병원체의 진단에 큰 도움을 줄 것으로 기대되었다. 결핵균의 진단방법중, 도말염색 방법은 감수성이 낮아서 문제가 되고 있으며, 배양은 감수성은 높으나 기간이 오래 걸려서 임상적으로 도움을 주지 못하는 경우가 많다. 이에 저자들은 Mycobacterium tuberculosis의 특이 단백질인 65 kD mycobacterial antigen을 encoding하는 2520 base pair DNA중, 383 base pair DNA를 이용한 PCR과 IS6110 fragment의 일부인 123 base pair DNA를 이용한 PCR을 시행하여, 이의 감수성과 특이도를 알아보고 폐결핵 환자의 객담을 검체로한 결핵의 조기진단 방법을 개발하고자 하였다. 방법 : M. tuberculosis (H37Rv, H37Ra), M. avium, M. intracellulare, M. scrofulaceum 균주와 환자의 객담에서 DNA를 추출하여, 383 base pair DNA 양끝의 20 base pair DNA primer (TB-1, -2)와 IS6110 fragment 일부의 DNA 양끝의 20 base pair DNA primer (Sal I-1, -2) 로 PCR을 시행하였으며, 전기 영동후 자외선 발광으로 확인하였다. 결과 : 1) Ethidium bromide 염색후 발광경하에서, Mycobacterium tuberculosis (H37Rv, H37Ra)와 Mycobacterium bovis는 TB-1, -2 primer와 Sal I-1, -2 primer를 이용한 PCR에서 모두 양성을 보였고 Mycobacter intracellulare와 Mycobacterium scrofulaceum은 TB-1, -2 primer를 이용한 PCR에서만 양성을 보였다. 2) Southern Blot 분석에는 두쌍의 primer 모두에서 Mycobacterium tuberculosis (H37Rv, H37Ra)와 Mycobactgerium bovis만이 양성을 나타내었으며 Mycobacterium intracellulare 와 Mycobacterium scrofulaceum은 음성을 나타내었다. 3) Mycobacterium tuberculosis (H37Rv)를 순차적으로 희석하여 시행한 PCR에서 두쌍의 primer 모두에서 Mycobacterium 균 1개체에 해 당되는 1 fg DNA까지 양성을 나타내었다. 4) 임상적으로 진단받은 결핵환자의 시행한, Sal I-1, -2 primer를 이용한 PCR에서 도말 검경 양성군의 객담 29예중 28예인 96.6%에서 양성을 나타내었으며, 도말 정경 음성-배양 양성군에서는 5예중 4예(80.0%), 그리고 도말 검경 음성-배양 음성군에서는 26예중 6예(23.1%)가 양성을 나타내었고 음성 대조군 검체 16예에서 2예(12.5%)에서 양성을 나타내였다. 결론 : 이상의 결과로, PCR은 객담에서의 결핵균의 진단에 있어, 배양과 견줄 수 있는 특이도와 예민도를 보이고 있어, 특정한 경우 진단에 도움을 줄 수 있을 것으로 기대되며, 추후 방법의 개선을 위한 연구가 계속 필요할 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제40권5호
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• pp.509-518
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• 1993

연구배경 : 결핵성 흉막 삼출은 아직까지도 우리나라에서 가장 흔한 흉막 삼출의 원인으로 생각되고 있으나, 조직학적 진단 방법을 포함한 기존의 진단 방법으로도 약 70%에서만 확진이 가능하다. 특히 고연령층이나 면역이 억제된 환자등에서 기존의 진단 방법으로 진단되지 않고, 여러가지 시험 치료에 반응하지 않는 흉막 삼출의 경우에는 임상적으로 어려움을 겪게 되며, 보다 예민한 진단 방법의 개발을 위해 여러 분야의 노력이 있어 왔다. PCR은 어느 특정한 DNA를 연속적으로 복제할 수 있어 검체내에 극미량으로 존재하고 있는 병원체의 진단에 이용되고 있으며, 결핵성 흉막염의 진단에도 도움을 줄것으로 기대되었다. 이에 저자들은 PCR을 이용한 결핵성 흉막염 진단의 유용성을 평가해 보고자 본 연구를 시행하였다. 방법 : 양성 및 음성 대조군으로 배양된 결핵균주및 비결핵성 Mycobacterium 균주를 사용하였고 연구 대상은 흉막 삼출 환자 53명이었다. 이중 결핵성 흉막염이 확진된 환자가 20명, 결핵성 흉막 삼출이 배제된 환자가 20명, 결핵이 확진되지 않았으나 임상적으로 결핵으로 의심하여 항결핵제제를 투여한 후 호전된 환자가 10명이었고, 항결핵제제 투여후에도 호전되지 않았으며 반복적인 진단 과정에서 결핵이 배제된 환자가 3명이었다. 대상 환자는 흉막 생검및 흉수의 ADA 활성도를 포함한 기존의 진단 방법과 PCR을 시행하였다. PCR의 target은 Mycobacterium tuberculosis의 특이성을 갖으며 한 개체내에 여러번 반복하여 존재한다고 알려져 있는 DNA인, IS6110 gene의 일부인 123 base pair DNA로 하였고, 그 결과를 기존의 진단 결과와 비교하였다. 결과: 1) 배양한 결핵균 및 비결핵성 Mycobacterium을 이용한 PCR의 강수성은 DNA 1 fg까지였고, Mycobacterium tuberculosis와 Mycobacterium bovis에만 특이적이었다. 2) 확진된 흉막액을 이용한 PCR의 감수성은 80.0%(16/20)였고, 특이성은 95.0%(19/20)였다. 3) 결핵이 확진되지 않았으나 임상적으로 결핵으로 의심하여 항결핵제제를 투여한 후 호전된 환자중 60.0%에서 PCR 양성을 나타내었다. 4) 결핵이 확진되지 않았으나 임상적으로 결핵으로 의심하여 항결핵제제를 투여한 환자중 호전되지 않고 추후 결핵이 배제된 환자 3명 모두에서 PCR 음성을 나타내었다. 5) 흉막액의 ADA 활성도는 확진된 결핵성 흉막염 환자군과 임상적으로 결핵이 의심되어 항결핵제 투여후 호전된 군이 여타군보다 유의하게 높았고, PCR 결과와 좋은 상관관계를 보였다. 결론 : PCR은 결핵성 흉막염의 진단에 매우 예민하고 특이적이었으며, ADA와의 좋은 상관 관계를 보였다. 따라서 PCR을 이용한 진단 방법을 추가함으로써 결핵성 흉막염의 진단에 도움을 줄 수 있을 것으로 사료되었다.

• Journal of information and communication convergence engineering
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• 제10권2호
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• pp.210-214
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• 2012

A microtiter well plate DNA hybridization method using Mycobacterium kansasii-specific rpoB DNA probe (kanp) were evaluated for the detection of M. kansasii from culture isolates. Among the 201 isolates tested by this method, 27 strains show positive results for M. kansasii, but the other 174 isolates were negative results for M. kansasii. This result was consistent with partial rpoB sequence analysis of M. kansasii and the result of biochemical tests. The negative strains by this DNA-DNA hybridization method were identified as Mycobacterium tuberculosis (159 strains), Mycobacterium avim (5 strains), Mycobacterium intracellulare (8 strains), and Mycobacterium flavescens (2 strain) by rpoB DNA sequence analysis. Due to high sensitivity and specificity of this test result, we suggest that DNA-DNA hybridization method using rpoB DNA probes of M. kansasii could be used for the rapid and convenient detection of M. kansasii.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.745-752
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• 2015

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from non-tuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/discrimination of this bacterium from clinical samples.

• 한국자기공명학회논문지
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• 제20권2호
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• pp.36-40
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• 2016

MRA1997 is a highly conserved protein from mycobacterial strains. However, no structural and functional information is associated with it. Thus, to obtain details about structure and function of this protein, we have utilized NMR spectroscopy. The recombinant MRA1997 was highly purified and its DNA binding mode was characterized. The tertiary structure of MRA1997 was modeled on the basis of our NMR chemical shift data combined with the webserver CS23D. The binding of MRA1997 with DNA was first monitored by electrophoresis mobility shift assays. The residues involved in DNA binding are identified using NMR chemical shift perturbation experiments. Based on our study, we suggest that MRA1997 interacts with DNA and may play an important role in Mycobacterium tuberculosis physiology.

• 대한의생명과학회지
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• 제10권2호
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• pp.163-169
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• 2004

Worldwide, tuberculosis remains one of the leading infectious diseases, accounting for nearly 3 million deaths and more than 8 million new cases annually. DNA typing of Mycobacterium tuberculosis is important for the control of tuberculosis, since it can be used to track transmission route of tuberculosis, source of internal laboratory contaminations, and to answer questions on the nature of tuberculosis infections such as reactivation or exogenous reinfection of disease. At present, IS6110-based RFLP is the choice of method for typing large numbers of clinical isolates of M. tuberculosis, since it has the highest resolution power. However, RFLP requires long time, high cost and qualified experts, so only reference level laboratories can use the RFLP technique. In order to have an optional molecular typing method suitable for the clinical settings, this study evaluated the use of one of PCR-based typing methods, IS6110-based outward PCR for typing clinical isolates of M. tuberculosis. In brief, the results from this study showed that IS6110-based RFLP is useful to discriminate diverse clinical isolates of M. tuberculosis as well as to identify clinical isolates that belong to the same family or cluster groups that have been previously classified by RFLP analysis. In addition, the banding profiles resulted from IS6110-based outward PCR seemed to represent genomic characteristics of M. tuberculosis, since strains belong to the K-family generated unique band that is not present in any other strains but present only in the genome of K-family strains. The IS6110-based outward PCR was also shown to be useful with DNAs isolated directly from liquid cultures indicating this method can be suitable for typing M. tuberculosis in clinical settings.

• Molecules and Cells
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• 제40권9호
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• pp.632-642
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• 2017

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.

• 대한의생명과학회지
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• 제15권4호
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• pp.295-300
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• 2009

Tuberculosis caused by Mycobacterium tuberculosis (MTB) still remains to be the most dreadful infectious disease affecting almost every country. In the present study, we developed a simple and rapid but accurate and sensitive assay method for detecting MTB using microplate hybridization assay. For this, a selective region of the rpoB gene was used to design PCR primers, and MTB and Mycobacterium genus-specific probe molecules. The specificity of the assay was confirmed using fifteen different mycobacterial reference strains and twelve different non-mycobacterial reference strains, and the sensitivity was determined to be 100 fg using genomic DNA of MTB reference strain, H37Rv. Subsequently, a total of 62 sputum samples with diverse smear scores and culture positive results were used to evaluate the kit performance. In brief, the specificity and the sensitivity of the assay were 100% and 98.4%, respectively.

• 한국동물위생학회지
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• 제34권4호
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• pp.321-331
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• 2011

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.