• Title/Summary/Keyword: RT-PCR detection

Search Result 475, Processing Time 0.028 seconds

Diagnosis Case of Viral Hemorrhagic Septicemia (VHS) in Adult Olive Flounder Paralichthys olivaceus (넙치(Paralichthys olivaceus) 성어의 viral hemorrhagic septicemia (VHS) 진단사례)

  • Kim, In-Woo;Cho, Mi Young;Lee, Han-Na;Han, Hyun Ja;Oh, Yun Kyeong;Lee, Soon Jeong;Jee, Bo Young;Myeong, Jeong-In;Won, Kyoung-Mi
    • Korean Journal of Fisheries and Aquatic Sciences
    • /
    • v.45 no.6
    • /
    • pp.666-674
    • /
    • 2012
  • We examined the cause of a disease outbreak in adult olive flounder Paralichthys olivaceus, which occurred at a Korean aquaculture farm in Korea in 2011. The principal signs included an expanded abdomen and congested liver, with persistent mortality (a little over two months). At the beginning of the outbreak, farm administrators misjudged the disease as bacterial in origin, because of the aforementioned signs, persistent mortality, and the detection of bacterial species, including Vibrio spp. and Streptococcus spp. Moreover, the detection of viral hemorrhagic septicemia virus (VHSV) by reverse trasnscription-PCR analysis was complicated by use of the VHS-VN primer set, which has been in general use recently, because it produced weak bands in some samples. Therefore, we recommend the use of at least two different primer sets in the diagnosis of VHSV. Our histopathological findings indicate that necrotizing myocarditis could be considered a pathogenic sign of VHSV infection.

Analysis of Bone Marrow Micrometastasis Using RT-PCR in Patients with Small Cell Lung Carcinoma (소세포 폐암에서 역전사 중합효소 연쇄반응(RT-PCR)을 이용한 골수 미세전이(naicronaetastasis)의 분석)

  • Kim, Tae-You;Park, Jong-Kook;Ryoo, Baek-Ryeol;Im, Yung-Hyuck;Kang, Yoon-Koo
    • Tuberculosis and Respiratory Diseases
    • /
    • v.47 no.6
    • /
    • pp.797-806
    • /
    • 1999
  • Background: About 20% of small cell lung cancer(SCLC) patients have bone marrow(EM) metastasis at the time of diagnosis and the remaining patients are also considered with micrometastasis. In an attempt to detect EM micrometastasis, we used cytokeratin(CK)-20 as a molecular marker, which is specific for epithelial cells. Method: A sensitive RT-PCR assay was used to compare CK-20 expression both in SCLC cell line H209 and normal leukocyte and to evaluate EM aspirates of 28 SCLC patients. Result: H209 cell line showed CK-20 expression but normal leukocyte did not, suggesting CK-20 expression is lung tissue-specific. Of 28 patients(11 limited disease, 17 extensive disease), only 2(1/11, 1/17) samples tested revealed positive signal for CK-20. Two patients with CK-20 expression had EM metastasis or multiple bone involvement during follow-up. Conclusion: Although circulating tumor cells were detected in EM of small portion of patients with bone metastasis, CK-20 doesn't seem to be a reliable marker for the detection of micrometastasis in SCLC. This study emphasizes that identification of more specific marker for micrometastsis is mandatory prior to clinical application.

  • PDF

Recently epidemiological survey of the viral diseases of broiler chickens in Jeonbuk province from 2005 to 2007 (최근 3년간 (2005-2007년) 전북지역 육계의 주요 바이러스성 질병 발생추이 분석)

  • Park, Jong-Beom;Cha, Se-Yeoun;Park, Young-Myoung;Zhao, Dan-Dan;Song, Hee-Jong;Jang, Hyung-Kwan
    • Korean Journal of Veterinary Service
    • /
    • v.31 no.1
    • /
    • pp.43-55
    • /
    • 2008
  • Recently, the major viral diseases, Newcastle disease (ND), infectious bronchitis (IB), low pathogenic avian influenza (LPAI), avian pneumovirus infection (APV), Marek's disease (MD) and infectious bursal disease (IBD), have led to huge economic losses in chicken industry of Korea. To evaluate prevalence of the major viral disease infections in broiler breeder and broiler farms, epidemiological survey has been conducted in Jeonbuk province from 2005 to 2007 by serological ELISA test for APV, PCR for MD, and RT-PCR for ND, IB, LPAI and IBD, respectively. A total of 424 cases was submitted to our laboratory for diagnosis of the major viral disease from broiler breeder and broiler farms in the above period. The diagnosed results were analysed for the detection rate of infections on basis of years, seasons and ages, respectively. This study was showed that the detection rates of ND and APV were considerably high for every years regardless of seasons and ages in both broiler breeder and commercial broiler. In comparison with detection rates of ND and APV, IB and LPAI were lower but detected around 10% for every years. Especially, detection rate of IB was significantly high in commercial broiler than in broiler breeder. Therefore, to minimize economic losses for broiler breeder and broiler farms, it will need for effective countermeasures to decrease detection rate of the viral respiratory diseases. Although the detection rates of MD and IBD were gradually decreased from 2005 to 2007 in both broiler breeder and commercial broiler, it will continually make an effort about disease control for increasing productivity in chicken industry.

Expression of Human Serum Albumin in Milk of Transgenic Mice Using Goat β-casein/Human Serum Albumin Fusion Gene

  • Wu, H.T.;Chou, C.K.;Huang, M.C.
    • Asian-Australasian Journal of Animal Sciences
    • /
    • v.17 no.6
    • /
    • pp.743-749
    • /
    • 2004
  • The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

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
    • /
    • 2003.10a
    • /
    • pp.68-68
    • /
    • 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

  • PDF

Production System of Virus-free Apple Plants Using Heat Treatment and Shoot Tip Culture (열처리와 경정배양을 이용한 바이러스 무병 사과 생산 시스템)

  • Lee, Gunsup;Kim, Jeong Hee;Kim, Hyun Ran;Shin, Il Sheob;Cho, Kang Hee;Kim, Se Hee;Shin, Juhee;Kim, Dae Hyun
    • Research in Plant Disease
    • /
    • v.19 no.4
    • /
    • pp.288-293
    • /
    • 2013
  • In worldwide, viral diseases of apple plants has caused the serious problems like reduced production and malformation of fruits. Also, the damages of apple plants by virus and/or viroid infection (Apple chlorotic leaf spot virus, Apple stem grooving virus, Apple mosaic virus, and Apple scar skin viroid) were reported in Korea. However there is few report about the protection approach against the infection by apple viruses. Therefore, this paper introduced the experimental protocol for the development of virus-free apple cultivars (Danhong, Hongan, Saenara, Summerdream). Apple plants were treated at $37^{\circ}C$ for 4 weeks and shoot tips were cultured in vitro. After heat treatment, the detection of apple viruses was performed by RT-PCR using virusspecific detection primers in new apple cultivars. With the heat treatments followed by in vitro shoot tip culture, the proportion of virus-free stocks of 'Danhong', 'Hongan', 'Saenara', and 'Summerdream' was 28%, 16%, 12%, and 12%, respectively. Taken together, this approach can be a good tool for production of virus-free apple stocks.

Detection of Mutations to Zidovudine in the pol Gene of Human Immunodeficiency Virus-1 by Direct Sequencing (인면역결핍 바이러스 pol 유전자 염기서열 결정에 의한 지도부딘 (ZDV) 내성 돌연변이의 탐지)

  • Cho, Young-Keol;Lee, Hee-Jung;Sung, Heung-Sup;Kim, Yoo-Kyum;Kim, Young-Bong;Lee, Yong-Jin;Kim, Mi-Jung;Kim, Dae-Ghon;Won, Young-Ho;Cho, Goon-Jae
    • The Journal of Korean Society of Virology
    • /
    • v.29 no.4
    • /
    • pp.271-281
    • /
    • 1999
  • The nested polymerase chain reaction (PCR) assay was used to determine the sequences of reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219 of human immunodeficiency virus-1 (HIV-1) pol gene. Template DNA was obtained from uncultured peripheral blood mononuclear cells from 27 Korean HIV-1 infected patients treated with ZDV and Korean red ginseng. The second PCRs were done for 2 separated regions (RT codons $13{\sim}98$ and $152{\sim}259$) with $5\;{\mu}l$ of the first PCR productNucleotide sequences were determined by direct sequencing. In the 27 patients, CD4+ cell count decreased from $230{\pm}117/{\mu}l$ to $152{\pm}162/{\mu}l$ for $46{\pm}26$ months (Mo), and actual duration of ZDV intake was $72{\pm}16$ Mo. In the 16 patients who had been treated with ZDV therapy ${\ge}25$ Mo, the incidences of 70R, 215F/Y, and 41L were 61%, 28% and 22%, respectively and those of 67N, 210W and 219Q were 17%. The incidences of 215F/Y were 6.7% for group ${\le}12$ Mo treatment, 22.7% for group with 13 to 24 Mo, and 27.8% for group ${\ge}25$ Mo. There was no mutation in 9 patients. It might be associated with the interruption of ZDV therapy for more than 6 months in 6 patients. This study shows that the detection of mutation could be useful prognostic marker with other clinical and virological data, and very low mutation rate is dectected compared to overseas reports.

  • PDF

Direct Stem Blot Immunoassay (DSBIA): A Rapid, Reliable and Economical Detection Technique Suitable for Testing Large Number of Barley Materials for Field Monitoring and Resistance Screening to Barley mild mosaic virus and Barley yellow mosaic virus

  • Jonson, Gilda;Park, Jong-Chul;Kim, Yang-Kil;Kim, Mi-Jung;Lee, Mi-Ja;Hyun, Jong-Nae;Kim, Jung-Gon
    • The Plant Pathology Journal
    • /
    • v.23 no.4
    • /
    • pp.260-265
    • /
    • 2007
  • Testing a large number of samples from field monitoring and routine indexing is cumbersome and the available virus detection tools were labor intensive and expensive. To circumvent these problems we established tissue blot immunoassay (TBIA) method an alternative detection tool to detect Barley mild mosaic virus (BaMMV) and Barley yellow mosaic virus (BaYMV) infection in the field and greenhouse inoculated plants for monitoring and routine indexing applications, respectively. Initially, leaf and stem were tested to determine suitable plant tissue for direct blotting on nitrocellulose membrane. The dilutions of antibodies were optimized for more efficient and economical purposes. Results showed that stem tissue was more suitable for direct blotting for it had no background that interferes in the reaction. Therefore, this technique was referred as direct stem blot immunoassay or DSBIA, in this study. Re-used diluted (1:1000) antiserum and conjugate up to 3 times with the addition of half strength amount of concentrated antibodies was more effective in detecting the virus. The virus blotted on the nitrocellulose membrane from stem tissues kept at room temperature for 3 days were still detectable. The efficiency of DSBIA and RT-PCR in detecting BaMMV and BaYMV were relatively comparable. Results further proved that DSBIA is a rapid, reliable and economical detection method suitable for monitoring BaMMV and BaYMV infection in the field and practical method in indexing large scale of barley materials for virus resistance screening.

Elimination of Grapevine fleck virus from infected grapevines 'Kyoho' through meristem-tip culture of dormant buds (휴면아 경정 배양법을 통한 포도 '거봉' 에서 Grapevine fleck virus의 제거)

  • Kim, Mi Young;Cho, Kang Hee;Chun, Jae An;Park, Seo Jun;Kim, Se Hee;Lee, Han Chan
    • Journal of Plant Biotechnology
    • /
    • v.44 no.4
    • /
    • pp.401-408
    • /
    • 2017
  • Herein, we report the meristem-tip culture from dormant buds of grape 'Kyoho' single-infected with Grapevine fleck virus (GFkV), which is phloem-limited and transmitted by graft inoculation. We produced GFkV-free shoots without thermo- or chemotherapy using meristem-tip explants approximately 0.3 mm (73 explants) and 0.8 mm long (five explants) including shoot apical meristem, 2-5 leaf primordia, and 1-4 uncommitted primordia from dormant buds of the infected woody cuttings (stored at $4^{\circ}C$). Explants were cultured on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 3.0 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-butyric acid (IBA). After 16 weeks of culture, shoot (10-mm long) regeneration frequency achieved from 0.3-mm explants was 4.1% and that obtained from 0.8-mm explants was 40.0%. Virus-free efficiency (expressed as the percentage of RT-PCR negative shoots regenerated) from 0.3- and 0.8-mm explants was 100% and 50%, respectively. Following in vitro multiplication, RT-PCR assays revealed identical results to assays of the first regenerated shoots. Our new methodological approach could be applied for eliminating other viruses in grapevines, as well as for producing virus-free plants in many other deciduous tree species, including fruit trees.

Epidemiology and Clinical manifestations of Enterovirus in Pediatric Inpatient in Incheon (인천지역 소아 입원 환자의 엔테로바이러스 역학과 혈청형에 따른 임상양상 비교)

  • Cho, Byoung Wook;Kwon, Seong Eun;Kwon, Mun Ju;Hur, Myong Je;Kim, Kyung Seon;Hong, Young Jin;Kim, Soon Ki;Kwon, Young Se;Kim, Dong Hyun
    • Pediatric Infection and Vaccine
    • /
    • v.23 no.1
    • /
    • pp.46-53
    • /
    • 2016
  • Purpose: Enterovirus (EV) infection in children can manifest various diseases from asymptomatic infection to nonspecific febrile illness, hand-foot-mouth disease, and aseptic meningitis. This study was aimed to investigate epidemiology and clinical significance of various genotypes of EV infections in pediatric inpatient. Methods: We collected the stool samples from the admitted pediatric patients in Inha University Hospital from March 2014 to March 2015. EV detection and genotype identification were performed by real-time RT-PCR and semi-nested RT-PCR. Phylogenetic trees were constructed by neighbor joining method. Results: A total of 400 samples were collected during study period and 112 patients (28%) were diagnosed with EV infections. The mean age of EV positive patients was 2.66 years (0.1-14) and sex ratio was 1.73:1. Genetic sequences of EVs were identified; coxsackievirus B5 (17, 15.2%), coxsackievirus A16 (13, 11.6%), enterovirus 71 (10, 8.9%), and coxsackievirus A2 (9, 8.0%). Nonspecific febrile illness (96, 86%) was the most common clinical manifestation and the duration of fever was 0-11 days (mean 3.1 days). Rash (44, 39%) and meningitis (43, 38%) were followed. Patients who were attending daycare center or had siblings accounted for 82.1%. Phylogenetic relationship tree revealed 6 distinct genogroups among 56 types of EVs. Conclusions: This study is the report of epidemiology, serotype distribution and clinical manifestations of children with EV infection in Incheon. This data will be helpful for further study about the epidemiology of EV infection in Korea.