• Title/Summary/Keyword: 역전사 중합효소 연쇄반응

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The Detection and Diagnosis Methods of Infectious Viroids caused Plant Diseases (식물체에 감염성 질병을 유발하는 바이로이드 검출 및 진단 방법)

  • Lee, Se Hee;Kim, Yang-Hoon;Ahn, Ji-Young
    • Journal of Life Science
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    • v.26 no.5
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    • pp.620-631
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    • 2016
  • Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.

GENE EXPRESSION ANALYSIS OF THE DENTAL PULP IN HEALTHY AND CARIES TEETH (치아 우식증에 따른 치수내 유전자 발현 변화에 관한 분석)

  • Oh, So-Hee;Kim, Jong-Soo
    • Journal of the korean academy of Pediatric Dentistry
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    • v.37 no.3
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    • pp.275-287
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    • 2010
  • Deep caries may induce pulpitis and the pulpal tissue interacts with microbial invasion. The immune response to protect the pulpal tissue can be mediated by cellular signal molecules produced by the pulpal cells. The understanding of these processes is important to find future therapeutic method for the diseased pulp. The pulp tissue from sound teeth was set as control group (n=30) and the pulp tissue from decayed teeth was set as test group (n=30). Total RNA was extracted from the pulp of each group and it was used for cDNA microarray and reverse transcriptase-polymerase chain reaction(RT-PCR). The expression of TGF-${\beta}1$ was studied by immunohistochemistry. The results were as follows: 1. cDNA microarray analysis identified 520 genes with 6-fold or greater difference in expression level with 143 genes more abundant in health and 377 genes more abundant in disease. 2. The RT-PCR analysis was done for randomly selected 14 genes and the results supported the result of cDNA microarray assay. 3. TGF-${\beta}1$ was highly expressed in the carious pulp and it was found in odontoblast by immunohistochemistry. In conclusion, many cytokines were found to be significantly changed their expression in the diseased pulp(/M/>1.6).

생쥐 초기 배아에서 Aquaporin 8과 9의 발현에 관한 연구

  • 신현상;계명찬;강수만;이성은;이지원;강한승;김문규
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.68-68
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    • 2003
  • Aquaporin은 막관통 통로 단백질(transmembrane channel protein)로서, 삼투압의 농도구배에 따라 세포막을 가로질러 물분자를 이동시키는 기능을 하고 있다. 포유류 초기배아에서 포배강 형성은 영양외배엽세포에서 $Na^+ / K^+$ATPase에 의한 이온 농도 구배가 형성되면 auqaporin에 의해 물이 포배강으로 유입되면서 이루어진다. 본 연구에서는 생쥐 초기배아에서 반정량적인 역전사 중합효소 연쇄반응 방법(semi-quantitative RT-PCR)과 실시간 역전사 중합효소 연쇄반응 방법(real-time RT-PCR)을 통하여 AQP8과 9의 mRNA발현을 조사하고 다중 면역형광현미경 방법(confocal immunofluorescence microscopy)을 통해 단백질 발현양상을 분석하였다. AQP8 mRNA는 상실기까지 발현되지 않다가 포배기에 이르러 발현되었고 AQP9 mRNA는 수정란에서부터 발현되어 포배기에는 유의할 정도로 증가하였다. 따라서 AQP8 mRNA는 배아유전자가 활성화되어 나타나는 것이고 AQP9 mRNA는 모계유전자 기원임을 알 수 있었다. AQP8 단백질은 상실배 단계까지 발현되지 않다가 포배시기에 영양외배엽세포사이의 접합면에 발현되었고 AQP9 단백질은 상실배 시기에 할구 사이의 인접 부위에서 강하게 발현되었다가 포배시기에는 세포간의 접합면에 약하게 발현하는 경향을 나타내었다. 실시간 역전사 중합효소 연쇄반응 방법으로 조사한 결과 포배에서 물과 글리세롤을 통과시키는 AQP9는 mRNA의 발현양이 AQP8보다 약 4배 정도 많았다. 또한 포배기에 이르러서야 물만을 통과시키는 AQP8의 발현이 나타나는 것을 보아 포배강 형성시 외부에서 영양외배엽을 통해 포배강으로 유입되는 물의 이동(trans- trophectodermal water movements)에 AQP9보다 AQP8이 더 중요하게 관여할 것으로 사료된다., K, Pb, Cd, Cr, Co, Cu, Ni)을 측정하였다. 실험 조건1의 결과로서 각 국의 유아용 일회용 기저귀의 중금속 함량은 거의 유사한 경향을 나타내었으며 Cr, Zn, Pb, Ni, Mn, Mg, Li, K는 detection limit(2 ppm) 이하였고, Cd, Fe, Co, Cu, Ca, Al, Sr는 검출되었지만 기준치 이하였다. 실험 조건2의 결과로서 측정 항목(Cr, Sb, Cd, Pb, Ni, Co, Cu)중 Cr, Cd, Ni, Cu는 detection limit(0.1 ppm) 이하였고, Sb, Pb, Co는 검출되었지만 기준치 이하였다.았다. 4%의 경우에는 8$0^{\circ}C$이하로 온도를 낮추는 것이 좋은 상태를 나타내었다. 이와 같은 결과는 일반적으로 화학적 레팅을 4%, 7%에서한 선행결과와 상당히 다른 결과이다.염 농도가 증가할수록 감소 현상을 보였다.X>, 75BG30은 8.6$\mu\textrm{m}$, 75BG40은 7.02$\mu\textrm{m}$로 나타났다. 따라서 경화제 양에 관계없이 10$\mu\textrm{m}$ 이하로 나타나, 경화제 10$m\ell$만으로 미세한 크기를 얻을 수 있음을 알 수 있다. 젤리 강도 변화에 따른 차이는 300BF는 78.09$\mu\textrm{m}$ 300BG는 56.32$\mu\textrm{m}$로, 75BF나 75BG에 비하여 현저히 증가하여, 젤라틴의 젤리 강도는 캡슐 제조 조건의 주요한 변수임을 알 수 있다.추출물 투여시 혈당강하 및 혈중콜레스테롤 강하가 나타났으며, 상엽복합추출물 투여와 운동을 병행시 이러한 감소 효과가 더 뚜렷하게 나타났다.교육의 적임자로 보는 시각이 비교적 높았고 약 1/2정도는 영양교육에 참여하겠다는 의지를 가지고 있을 뿐만 아니라 실제로 영양지도를

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Biological Evaluation of Bone Marrow-Derived Stem Cells onto Different Wettability by RT-PCR (역전사 중합효소 연쇄반응을 이용한 표면 적심성에 따른 골수유래 줄기세포의 생물학적 평가)

  • 김은정;박종수;김문석;조선행;이종문;이해방;강길선
    • Polymer(Korea)
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    • v.28 no.3
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    • pp.218-224
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    • 2004
  • The adhesion and proliferation of mammalian cells on polymeric biomaterials depend on the surface characteristics such as wettability, chemistry, charge and roughness. In order to recognize the correlation between the adhesion and proliferation of human bone marrow derived stem cells (BMSCs) and surface property, radio frequency generated plasma treatment on low density polyethylene (LDPE) has been carried out. The modified LDPE surfaces were characterized by measuring the static water contact angle. The adhesion and proliferation of cells on LDPE films were characterized by cell counting and reverse transcription-polymerase chain reaction (RT-PCR). The water contact angle of the film surface decreased with plasma treatment time. Proto-oncogenes (c-myc, c-fos) and tumor suppressor gene (p153) showed maximum expression with contact angle of 60 ∼ 70$^{\circ}$ range of LDPE film. By cell counting, we confirmed that the rate of cell proliferation appeared the higher on the film surface of the contact angle of 60∼70$^{\circ}$ We concluded that the surface wettability is an important role for the growth and differentiation of BMSCs.

Development and Evaluation of a SYBR Green Real-time PCR Assay for Canine Cytokine Gene Expression (SYBR Green 실시간 역전사 중합효소연쇄반응을 이용한 개 싸이토카인 유전자 발현의 정량)

  • Yu, Do-Hyeon;Ihn, Dong-Chul;Park, Chul;Park, Jin-Ho
    • Journal of Veterinary Clinics
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    • v.27 no.5
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    • pp.508-513
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    • 2010
  • Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

A Retrospective Analysis of Use in Hospitalized Children with Upper Respiratory Tract Infection (상기도 감염으로 입원한 소아환자에서 항생제 사용에 대한 후향적 분석)

  • Jung, Minyoung;Park, Ji Hyun;Oh, Chi Eun
    • Pediatric Infection and Vaccine
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    • v.24 no.2
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    • pp.87-94
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    • 2017
  • Purpose: The inappropriate prescription of antibiotics in children with upper respiratory tract infection (URTI) is common. This study evaluated the factors that influence antibiotics use in hospitalized children with viral URTI confirmed by reverse transcriptase-polymerase chain reaction (RTPCR) assay. Methods: The medical records of admitted patients who performed RT-PCR assay for respiratory virus pathogens from January 2013 to November 2014 were examined. The demographic and clinical features were compared between patients who were administered antibiotics at admission and those who were not. We also investigated differences between children who continued antibiotics and those who stopped antibiotics after a viral pathogen was identified. Results: In the total 393 inpatients, the median age was 23 months (interquartile range, 13 to 41.3 months). Antimicrobial agents were prescribed in 79 patients (20.1%) at admission. Patients with acute otitis media (AOM) had higher rates of antibiotics prescription than those without AOM (48.1% vs. 2.2%, P<0.001), with an adjusted odds ratio of 91.1 (95% confidence interval, 30.5 to 271.7). Level of high-sensitivity C-reactive protein and the proportion of acute rhinosinusitis were also significantly associated with antibiotics use (P<0.001). Among the 44 patients with viruses identified using the RT-PCR method during hospitalization, antibiotic use was continued in 28 patients (63.6%). AOM was statistically associated with continued antibiotic use in the patients (P=0.002). Conclusions: Although the respiratory virus responsible for URTI etiology is identified, clinicians might not discontinue antibiotics if AOM is accompanying. Therefore, careful diagnosis and management of AOM could be a strategy to reduce unjustified antibiotic prescriptions for children with URTI.

The Effect of ${\Delta}^{12}PGJ2$ and $PPAR{\Gamma}$ Agonist on the Proliferation and Differentiation of Osteoblast ((${\Delta}^{12}PGJ_2$ 및 PPAR 감마 길항체가 조골세포의 증식 및 분화에 미치는 효과)

  • Heo, Jeong-Mi;Kim, Kyoung-Wha;Chung, Kyoung-Wook;Lee, Hye-Joon;Rhyu, In-Chul;Ku, Young;Chung, Chong-Pyoung
    • Journal of Periodontal and Implant Science
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    • v.35 no.1
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    • pp.133-152
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    • 2005
  • 1. 목적 Prostaglandin은 치주질환과 관련된 국소적 골 대사에 중요한 역할을 한다. ${\Delta}^{12}PGJ_2$는 생체 내에서 혈장의 존재 하에 형성되는 천연 $PGD_2$ 대사산물이며 peroxisome- proliferator에 의해 활성화되는 감마 수용체 (PPAR ${\Gamma}$)에 대해 높은 친화성을 갖는 리간드로서 핵 수용체군에 속하는 전사조절인자이다. 이 연구의 목적은 골화 과정에서 ${\Delta}^{12}PGJ_2$의 역할을 규명하기 위해, 조골세포주의 증식과 분화에 미치는 영향과 그에 관련된 세포기전을 조사하는 데에 있다. 2. 방법 인간 골육종세포주인 Saos-2 (ATCC.HTB 85)와 쥐의 조골세포주 (MC3T3-E1)를 배양한 후 실험군에 농도가 각각 $10^{-5}$, $10^{-6}$, $10^{-7}$, $10^{-8}$, $10^{-9}$ 몰인 ${\Delta}^{12}PGJ_2$와 ciglitazone (합성 PPAR 감마 길항체)를 첨가하였다. 조골세포에서 PPAR 감마의 발현을 관찰하기 위해 역전사효소-중합효소연쇄반응(RT-PCR)을 특정한 primer를 이용하여 시행하였다. 세포 증식은 1일, 2일, 3 일째에 MIT 분석법으로 측정하였고, 2 일째에 알칼리성 인산효소 (ALPase) 생산을 측정하였다. 위의 결과에서 얻은 적정한 농도에서 다양한 조골세포 분화의 표지자들-제 1 형 교원질, 알칼리성 인산효소, osteopontin 및 bone sialoprotein-에 대한 간이 정량적 역전사효소-중합효소연쇄반응 (semiquantitative RT-PCR)을 실시하였으며 골결절 형성에 대한 효과를 알아보고자 석회화 분석도 시행하였다. 3. 결과 ${\Delta}^{12}PGJ_2$와 ciglitazone 모두 Saos-2 세포주의 증식을 촉진시켰다 .$10^{-8}$ 몰의 ${\Delta}^{12}PGJ_2$$10^{-6}$몰의 ciglitazone을 첨가한 실험군을 대조군과 비교했을 때, 시간에 비례하여 세포 증식률이 증가되었다. 알칼리성 인산효소의 활성화 검사에서도 증식률에서와 유사한 결과를 보여주었다. 간이 정량적 RT-PCR에서는 ${\Delta}^{12}PGJ_2$로 처리한 군의 경우 제 1 형 교원질, 알칼리성 인산효소, osteopontin, 그리고 bone sialoprotein의 상대적 mRNA 수준이 유의하게 높았다. 석회화 분석에서는 MC3T3-E1 세포를 $10^{-6}$ 몰의 ${\Delta}^{12}PGJ_2$로 처리한 군과 $10^{-5}$ 몰의 ciglitazone으로 처리한 군에서 현저한 골결절 형성을 보였다. 이러한 결과들은 ${\Delta}^{12}PGJ_2$가 유용한 골 유도물질이 될 수 있으며 또한 그 작용기전이 PPAR 감마-의존형 경로와 연관되어 있음을 보여준다.

Analysis of Gene Mutation and Expression Level of Follicle Stimulating Hormone Receptor in Premature Ovarian Failure(POE) Patients (조기 난소 부전증(Premature Ovarian Failure, POF) 환자에서 난포 자극 호르몬 수용체 유전자 변이 및 발현 양상에 대한 분석)

  • 김정욱;염혜원;이형송;송견지;천강우;박용석;김계현
    • Development and Reproduction
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    • v.4 no.1
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    • pp.61-66
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    • 2000
  • This study was investigated to analyze the inactivating point mutation and expression level of follicle-stimulating hormone(FSH) receptor mRNA. In first experiment, we analyzed the point mutation. Peripheral blood was collected from each patient. To screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 10 patients. No inactivating point mutation of FSH receptor gene was identified in women with premature ovarian failure. To analyze the expression level of FSH receptor, mRNA expressions were examined by RT-PCR method using specific primers for the FSH receptor. The amount of FSH receptor mRNA expressed in POF patients was lower than that in the control group. But it was not significantly different. These finding suggests that lower expression of FSH receptor in premature ovarian failure patients might be the cause of the low response to the gonadotropin during the hyperstimulation in IVF-ET cycles.

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Detection of Cucumber Mosaic Virus by RT-PCR Using a Simple and Rapid Crude Sap Extraction Method (간이 조즙액 추출법을 이용한 RT-PCR 방법에 의한 오이 모자이크 바이러스의 검정)

  • 이상용;홍진성;이진상;최장경
    • Korean Journal Plant Pathology
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    • v.12 no.4
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    • pp.432-436
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    • 1996
  • 역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 담배(Nicotiana glutinosa)에 증식시킨 7종의 오이 모자이크 바이러스(CMV)를 검정하였다. RT-PCR을 위한 간단하고 신속한 바이러스 핵산의 조즙액 추출법이 개발되었으며, CMV 외피단백질 유전자 부위를 기초로 하여 제작한 20개의 염기로 구성된 primer를 사용하여 RT-PCR을 실시한 결과, 약 490 염기쌍의 DNA 단편들이 이병식물의 조즙액으로부터 증폭되었다. EcoRI 및 MspI을 이용한 RT-PCR 산물의 분석에 의하여, 공시한 7종의 바이러스는 모두 CMV subgroup I으로 동정되었다. Ouchterlony 한천젤 이중 확산법을 이용한 항혈청 검정에서도 7종의 바이러스 모두 CMV-Y의 항혈청과 단일의 침강선을 형성하였다.

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Identification of Introduced Gene and Its Expression and Gene Stability Assessment for Event Selection of Genetically Modified Plant toward Approval: Cucumber Mosaic Virus Resistant Hot Pepper (상업용 유전자 변형작물 이벤트 선발을 위한 도입유전자 확인, 발현 및 세대간 안정성 평가 : 오이모자이크바이러스 저항성 GM 고추)

  • Kang, Seung-Won;Han, Bal-Kum;Lee, Tae-Ho;Kim, Eun-Ji;Lee, Gung-Pyo
    • Horticultural Science & Technology
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    • v.30 no.2
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    • pp.192-200
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    • 2012
  • For the development of genetically modified plants, it is important to verify various factors which potentially affect the risk assessment as well as to establish an experimental program to produce scientific and reliable data. However, it is a time and cost consuming process to develop GM plants as well as to prepare scientific and convincible data for government's approval. Therefore, using the transgenic hot pepper tolerant to a new CMV pathotype, we attempted to suggest few methodological procedures, such as probe saturation for southern blot analysis and RT-PCR and ELISA for expression analysis, for identification and stability evaluation of inserted gene in genetically modified plant which are required for submission for approval. Ten partially overlapped probes covering full length of inserted gene were produced. We could identify that the inserted gene was stacked as a single copy as well as no partial element existed. Also, we could identify the stability of the inserted gene stacked in hot pepper using probe saturation. In the expression analysis with RT-PCR and ELISA, we also could provide the stable expression of transcript and proteins in leaves and placenta and pericarp of fruits of the CMV-resistant hot pepper.