• 대한임상검사과학회지
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• 제39권1호
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• pp.49-55
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• 2007

HCV는 single stranded RNA 바이러스로서 감염 시에는 만성간염 및 간경화 간암으로 진행될 수 있는 가능성이 높다. HCV는 6종의 주된 genotype과 그에 따른 많은 종류의 subtype이 보고되고 있으며, 세계 각 지역별로 그 분포는 매우 다양하다. 여러 가지 HCV genotype 중에서 1b 형에 감염되었을 경우 간경화나 간암으로 진행할 가능성이 높으며 치료효과도 떨어진다는 보고가 있어, 최근 HCV 환자의 치료에 있어서 HCV 바이러스 정량검사와 함께 HCV genotyping 검사의 임상적 활용이 높아지고 있다. 본 연구에서는 PCR-direct sequencing을 이용한 HCV genotyping 검사방법을 이용하여, 한국인 만성 HCV 간염환자에서 HCV genotype의 분포를 조사하였다. 검체로는 232명의 한국인 만성간염환자의 혈청을 사용하였으며, HCV 5'UTR 영역에서 선택한 2쌍의 primer로 nested PCR을 실시하였다. 증폭된 PCR산물 (215 bps)은 2% agrose gel로 전기영동을 하고 sequencing을 실시한 후 GeneBank의 BLAST 프로그램을 사용하여 HCV genotype을 분석하였다. HCV genotyping을 실시한 232명에서 5종류의 genotype, HCV 1b, 2a, 2b, 2c, 3a, 이 발견되었으며, HCV genotype 4, 5, 6 은 검출되지 않았다. 발견된 HCV genotype 중에서 HCV 1b의 검출률이 53.9%로 가장 높았고, 다음은 HCV 2a가 35.8%로 높게 나타나, 위 두 가지 HCV genotype을 합하면 거의 90%였다. 다음으로 HCV genotype 2b가 3.9%, 3a가 3.4% 그리고 2c가 3.0%의 순서로 검출되었다. 본 결과는 한국인 만성 HCV간염 환자의 치료 및 예후관리에 참고가 될 것으로 사료된다. 또한 PCR-direct sequencing을 이용한 HCV genotyping 검사는 간편하고 분명하게 결과를 판독할 수 있어 임상실험실에서 유용하게 사용될 수 있을 것으로 판단된다.

• Molecules and Cells
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• 제26권2호
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• pp.212-215
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• 2008

The GC-rich discriminator sequence between the -10 region and the transcription start site of the rnpB promoter is responsible for stringent control of M1 RNA synthesis. The rnpB promoter also contains a G nucleotide at the previously identified transcription start site. In this study, we examined by mutagenesis of G to A whether this +1G nucleotide is involved in the stringent response. We found that the change did not alter the stringent response. Since the +1 mutation might alter transcription initiation, we compared the transcription start sites of the wt and mutant promoters by primer extension analysis. Surprisingly, we found that wild type rnpB transcription starts at both the +1G position (70%) and the -1C position (30%), and that the +1A mutation led to transcription initiation exclusively at the -1C position. We also generated two transversion mutations at the -1 position, both of which led to transcription starting exclusively at that position. The -1G mutant promoter gave a stringent signal similar to the wild-type, whereas the -1A mutant generated a significantly less stringent signal. Base on these results, we propose that a short sequence, up to 7 bp downstream of the -10 region, is involved in the stringent response of the rnpB promoter.

• The Plant Pathology Journal
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• 제31권3호
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• pp.219-225
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• 2015

The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

• Journal of Microbiology and Biotechnology
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• 제21권4호
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• pp.333-340
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• 2011

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P<0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.

• Applied Biological Chemistry
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• 제44권3호
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• pp.153-161
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• 2001

Ecdysteroid 생합성에 관련된 식물 유전자를 분리할 목적으로 ecdysteroid 생성능이 확인된 쇠무릎(Achyranthes japonica Nakai)으로부터 RNA를 분리하고, ecdysteroid 생합성에 관여한다고 알려진 cytochrome P45O family 유전자 4개와 곤충의 ecdysone 20-hydroxylase 유전자의 다중정렬 분석결과를 토대로 상동성이 높은 부위의 염기서열에 상응하는 degenerate primer를 사용하여 RT-PCR을 행하고, 고유한 염기서열을 가진 partical cDNA clone 14개를 선발했다. 기존에 ecdysteroid 생합성에 관여한다고 알려진 cytochrome P450 family 유전자들과의 염기 및 아미노산 서열의 상동성을 분석한 결과, 선발된 cDNA 중 6개가 cytochrome P45O fumily 유전자들과 상동성이 있는 것으로 나타났다. 이 중에서 4개의 clone은 곤충의 ecdysone 20-hydroxylase 유전자와도 상동성이 높아 ecdysteroid 생합성에 관련된 유전자로 추정되었다.

• Journal of Dairy Science and Biotechnology
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• 제24권1호
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• pp.19-28
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• 2006

유아 분변으로부터 분리한 52 균주는 그람 양성균인 Listeria monocytogenes KCCM 40307$^T$에 대하여 항균활성이 있었고 그 중에서도 선별 균주 A24의 항균활성이 45% 이상으로 가장 높았다. Bifidobacterium longum A24의 생육 및 항균 물질 생산을 검토하였을 때 균체의 생육은 28시간 배양 시 최고에 도달하였고 항균 활성은 36시 간 배양 시 최고를 나타내었다. 선별균주 A24는 16S rRNA-based molecular typing 결과Bifidobacterium 속 균주임을 확인할 수 있었고 형태학적 ${\cdot}$ 생화학적인 방법으로 검토하여 보았을 때 Bifidobacterium longum으로 판단되었으며, 16s rDNA sequencing 결과 최종적으로 Bifidobacterium longum로 동정 되었으며 이것을 Bifidobacterium longum A24로 명명하였다. Bifidobacterium longum A24의 항균 활성 물질은 균체의 생육이 가장 좋은 28시간 배양에서가 아니라 그보다 늦은 36시간 배양에서 최고를 나타내었고 그 이후로는 활성이 감소하는 경향을 보였다. 이것은 Bifidobacterium longum A24이 생성하는 항균 활성 물질이 bacteriocin과 같은 2차 대사 산물임을 암시하는 결과로 해석된다.

• 대한한의학회지
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• 제21권4호
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• pp.16-25
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• 2000

Objective : Acupuncture and Radix Astragali aqua-acupuncture stimuli have long been used to cure human diseases. However, the exact physiological and biochemical mechanisms involved remain undiscovered. Thus, many attempts have been made to show the scientific mechanisms involved. The effects of acupuncture and Radix Astragali aqua-acupuncture, which was known to date, as follow; effective circulation of body blood system and proliferation of leucocytes. Methods : In this study, we have applied acupuncture and Radix Astragali aqua-acupuncture stimuli to mouse on Sinsuhyul, a stimulative point of oriental medicine, to see effects on the expression of cytokine $IL-1{\beta}$. Mice were treated with lipopolysaccharide(LPS) for inflammation induction and then reverse transcriptase-polymerase chain reaction (RT-PCR) using each primer set were performed to trace the amounts of mRNA. Results : 1. $IL-1{\beta}$ was not expressed in LPS-nontreated mice at 15 to 60 min after acupuncture-stimuli. However, expression occurred after 3hrs. 2. $IL-1{\beta}$ was specifically expressed in LPS-treated mice at 30 min after acupuncture-stimuli. 3. $IL-1{\beta}$ was expressed in LPS-nontreated mice at 30 min after Radix Astragali aqua-acupuncture stimuli, however, not expressed at 60, 180 min. 4. $IL-1{\beta}$ was gradually expressed in LPS-treated mice at 15 to 180 min after Radix Astragali aqua-acupuncture stimuli. Conclusions : $IL-1{\beta}$ in LPS-treated mice was more effective than that of LPS-nontreated mice. We are now in the process of elucidating the immunological action mechanism of acupuncture and Radix Astragali aqua-acupuncture stimuli. And cytokine $IL-1{\beta}$ can be used not only as a basis of the effects of acupuncture and Radix Astragali aqua-acupuncture but also as a diagnosis guide through the immunological actions of those.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2153-2159
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• 2015

Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation — only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

• 한국어병학회지
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• 제18권2호
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• pp.157-165
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• 2005

2005년 여름에 국내에서 사육되는 가물치에서 대량폐사를 수반하는 새로운 질병이 발견되었다. 감염 어류는 복부팽만과 항문 주위 출혈 이외의 특이한 외부 증상을 나타내지 않았으나 복부를 절개하면 많은 수의 백색 결절이 간, 비장 및 신장을 포함한 내부 장기에서 관찰되었다. 특히 내부 장기의 조직병리학적 관찰에서 granuloma가 관찰되었다. 이들 감염 어류에서 Gram 양성의 사상균이 분리되었다. 분리균은 Nocardia spp.와 non-Nocardia 균의 16S rRNA 염기 분석에 기초한 Nocardia 특이 primer를 사용한 PCR 방법으로 확인하였다. 이것은 어류에서 발생한 Nocardia 감염증의 국내 첫 사례이다.

• 한국산학기술학회논문지
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• 제14권6호
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• pp.3114-3119
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• 2013

망그로브 생태계는 수생태계로 유입되는 중금속을 받아들이는 기능을 가지고 있다. 중금속에 오염된 퇴적물에 노출됨에도 불구하고 망그로브는 중금속에 내성을 가지고 있다. 이 연구에서 우리는 망그로브로부터 중금속 저항성 관련 유전자를 클로닝하고, 중금속 노출에 유전자 발현 변화를 분석하였다. 미크로네시아 축라군의 웨노섬에서 채취한 Rhizophora stylosa의 잎과 뿌리조직으로부터 CTAB 방법을 이용하여 RNA를 분리하였고, gene specific primers를 이용하여 phytochelatin synthase 1(PCS1) 유전자를 클로닝하였다. R. stylosa 태생종자를 100 ppb의 Cd과 10 ppb의 Cu에 노출하였을 때 각각 1.91배 및 2.72배 발현이 증가하였다. 이러한 결과는 PCS1 유전자의 발현분석이 망그로브 생태계의 건강성을 평가하기 위한 좋은 도구가 될 수 있음을 나타낸다.