• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.287-293
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• 2001

A fast and easy PCR-SSCP method was developed and assessed for the early detection of rifampin-resistant Mycobacterium leprae in skin biopsy samples from Korean leprosy patients. The 190 bp of the rpoB gene, in which mutation is known to cause resistance to rifampin, was amplified by PCR and then analyzed by SSCP and DNA sequencing, All PCR products showing mobility shift on PCR-SSCP contained mutations, demonstrating that this method can be used for an early diagnositic method to detect a putative rifampin-resistant M. leprae strain. DNA sequence analysis revealed that 19 of 34 patient samples contained M. leprae strains with missense mutations in the rpoB gene: five were the same mutations previously reported to cause rifampin resistance and eight were the new type of mutatios that likely cause rifampin resistance. These newly identified dmutations, whose all five cytosine bases of four amino acids were substitued with thymine, were found at different sites from those reported in Mycobacterium tuberculosis or M. leprae. Therefore, they may provide additional clues to understand the molecular biological basis on the rifampin resistance of M. leprae.

• Journal of the Korean Wood Science and Technology
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• 제46권4호
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• pp.402-414
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• 2018

Species identification of wood provides important information for archaeology, restoration of cultural assets, preventing illegal logging, and more. Wood species are usually identified based on their anatomical features with the use of a microscope. However, this method may not be able to distinguish between anatomically similar species or subspecies. To overcome this problem, wood species need to be identified at the molecular level using DNA sequencing. However, unlike living plant cells, wood is difficult to pulverize using a mortar, and DNA extraction from dried wood is challenging. To solve these problems, we propose a pretreatment method in which wood is pulverized using 60-grit sandpaper and hydrated with water for 2 days. Using this method, we were able to stably amplify the rpoB gene from the extracted DNA of Pinus rigida. In addition, sequence analysis of the rpoB gene revealed six single nucleotide polymorphisms (SNPs), which classified the rpoB sequences in the genus Pinus into five groups. Our data indicate that although these SNPs were not suitable for species identification, they can potentially be used to determine the origin of different wood subspecies or individual samples of wood.

• 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.

• Journal of Microbiology
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• 제35권3호
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• pp.177-180
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• 1997

The purpose of this study was to evaluate the efficiency of Line Probe Assay (LiPA) in detecting the rpoB gene mutation of clinically isolated Mycobacterium tuberculosis (MTB) and to compare the level of resistance to the various rifamycins with their mutation sites. The mutation in the rpoB gene was found in 84 (97.6%) out of 86 rifampicin (RMP) resistant strains as determined by LiPA. No mutation was observed in 2 RMP resistant strains and in any of 38 RMP susceptible strains tested. Only one of 3 strains with .DELTA.5/R5, one of 2 strains with .DELTA.3, and one of 3 strains with .DELTA.2/R2 LiPA profile showed a slightly lower level of resistance to the rifapentine than the other strains. Although we could not find correlations between mutation sites in the rpoB gene and the level of susceptibility to the various rifamycins, the LiPA is recommended as a fast screening tool for detection of RMP resistant MTB.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.258-265
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• 2004

Numerous genes of Escherichia coli have been shown to growth phase-dependent expression throughout growth. The global patterns of growth phase-dependent gene expression of E. coli throughout growth using oligonucleotide microarrays containing a nearly complete set of 4,289 annotated open reading frames. To determine the change of gene expression throughout growth, we compared RNAs taken from timecourses with common reference RNA, which is combined with equal amount of RNA pooled from each time point. The hierarchical clustering of the conditions in accordance with timecourse expression revealed that growth phases were clustered into four classes, consistent with known physiological growth status. We analyzed the differences of expression levels at genome level in both exponential and stationary growth phase cultures. Statistical analysis showed that 213 genes are shown to, growth phase-dependent expression. We also analyzed the expression of 256 known operons and 208 regulatory genes. To assess the global impact of RpoS, we identified 193 genes coregulated with rpoS and their expression levels were examined in the isogenic rpoS mutant. The results revealed that 99 of 193 were novel RpoS-dependent stationary phase-induced genes and the majority of those are functionally unknown. Our data provide that global changes and adjustments of gene expression are coordinately regulated by growth transition in E. coli.

• Journal of Microbiology and Biotechnology
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• 제18권11호
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• 대한미생물학회지
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• 제34권5호
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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.

• 한국어병학회지
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• 제30권2호
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• pp.79-88
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• 2017

현재 양식장에서는 Aeromonad를 비롯한 다양한 병원균에 의한 전염병으로 인해 많은 경제적 손실을 겪고 있다. 연어과 어류뿐만 아니라 담수 및 해수어류에도 치명적인 감염을 야기하는 Aeromonas 종은 적어도 26종 이상이 보고되어왔으며, 전염병을 유발하는 유비쿼터스 세균이다. Aeromonas 종을 확인하기 위해 16S rDNA 및 하우스 키핑 유전자의 핵산 서열을 기반으로 한 분자생물학적 기술이 사용될 수 있다. 본 연구에서 Aeromonas 종은 강원도 16개 양식장의 연어과 어류로부터 분리되었으며 Aeromonad의 16S rDNA와 하우스 키핑 유전자의 서열, 즉 RNA polymerase sigma factor ${\sigma}^{70}$ (rpoD) 또는 DNA gyrase subunit B (gyrB)를 기반으로 계통 발생 학적으로 동정했다. 그 결과 대서양 연어 (Salmo salar), 은연어 (Oncorhynchus keta), 산천어 (Oncorhynchus masou masou), 무지개송어 (Oncorhynchus mykiss)에서 96 개의 균주가 수집되었으며, 36개의 균주가 16S rDNA 분석에 의해 Aeromonas 속으로 확인되었다. 확인된 Aeromonas 속 균주는 rpoD 또는 gyrB 유전자 서열을 기반으로 추가 분석되어 Aeromonas salmonicida (24 균주), A. sobria (10 균주), A. media (1 균주) 및 A. popoffii (1 균주)로 검출되었으며, 이 것은 Aeromonas salmonicida가 강원도의 연어과 어류에서 주요 감염균임을 나타낸다. 또 하우스 키핑 유전자의 서열에 기초한 Aeromonas 종의 계통발생학적 동정은 16S rDNA 서열보다 더 정확하다는 것이 또한 증명되었다.

• 한국식품과학회지
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• 제40권1호
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• pp.96-100
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• 2008

향신성 식용유는 에너지원이나 대사조절물질로서뿐 아니라 식품의 맛이나 향 또는 포만감을 결정하는 지방이다. 본 연구는 0.12% 콜레스테롤 식이를 한 흰쥐에서 대두유를 대조로 하여 향신성 식용유(고추종실유, 산초유)에 의한 지질농도의 조절효과를 평가하였다. 사료중 n-6/n-3 지방산의 비는 8.8:1.2:70.1 (대두유:산초유:고추종실유)로 큰 차이가 있었다. 혈청콜레스테롤 농도는 고추종실유 섭취군에서 다른 군보다 높았으나, HDL-cholesterol/totalcholesterol 비는 고추종실유 섭취군에서 낮았다. 혈청중성지질 농도는 다른 군에 비하여 산초유 섭취군에서 낮았다. 반면에 간장 콜레스테롤 농도는 고추종실유 섭취군보다 산초유 섭취군에서 높았다. 이러한 응답은 식이지방에 의한 혈청 콜레스테롤 농도의 응답은 조직간 콜레스테롤 분배의 차이에 기인되는 듯하다. 결론적으로 본 연구는 콜레스테롤 함유식이를 한 흰쥐에서 고추종실유는 대두유에 비하여 콜레스테롤상승효과를 보여준 반면, 산초유는 중간적 효과를 보여주었다.

• 한국식품영양과학회지
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• 제18권3호
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• pp.307-315
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• 1989

${\omega}3$계와 ${\omega}6$계 PUFA의 함유비율을 달리했을때 흰쥐장기의 지질대사 및 혈청 lipoprotein 조성에 미치는 영향을 알아보고자 실험한 결과를 요약하면 아래와 같다. 뇌 조직중의 총cholesterol농도는 전 식이군 간에 유의성은 없으나 ${\omega}6$계 linoleic acid 함유율이 높은 고추씨기름 10%단독 식이군에서 높은 함량을 보이는 반면 ${\omega}3$계 linolenic acid 함유율이 높은 들깨유 식이군 및 혼합 식이군에서 감소하는 경향을 보였다. 한편 중성지방 및 인지질 농도는 고추종자유 함유율이 높은 식이군에서 서서히 증가하지만 들깨유 함유량이 많아질수록 점차 감소하였다. 신장 조직중의 총cholesterol농도는 전 군간에 유의성이 없었으며, 중성지방 농도는 ${\omega}6$계 linoleic acid함유 비율이 높은 고추종자유 식이군이 가장 높았고 혼합 식이군에서 낮았다. 인지질 농도는 10% 고추종자유 식이군에서 현저히 낮았다. 고환 조직중의 총cholesterol농도는 혼합 식이군 및 들깨유 식이군에서 낮았으며, 중성지방 농도는 들깨유 식이군에서 상승하였고 혼합 식이군에서 감소하였다. 인지질 농도는 5군에서 낮았다. 혈청 lipoprotein pattern에서 HDL은 들깨유 함량이 많은 식이군(1,2,3군)에서 상승된 반면 고추씨기름 식이군에서는 가장 낮았으며, LDL은 들깨유 비율이 높아질수록 점차로 낮아졌다.