• 식물병연구
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• 제16권2호
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• pp.121-124
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• 2010

박과작물에 발생하는 종자전염 바이러스 3종(CGMMV, KGMMV, ZGMMV)에 대한 검출을 위해 IC-RT-PCR의 적용 가능성을 시험하였다. IC-RT-PCR을 이용하여 감염 종자와 잎으로 부터 바이러스의 특이적인 검출이 가능하였으며, 검출 민감도는 ELISA 보다 100배 이상 높았다. 또한 반응을 마친 ELISA 마이크로플레이트에 남아있는 항원을 IC-RT-PCR의 template로 사용할 경우 ELISA 결과를 곧바로 확인할 수 있었다. 따라서 IC-RT-PCR에 의한 본 검정방법은 박과작물의 대규모 포장검사 및 종자검사에 편리하게 사용될 수 있을 것으로 기대된다.

• The Plant Pathology Journal
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• 제22권2호
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• pp.164-167
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• 2006

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

• 한국버섯학회지
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• 제1권1호
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• pp.28-33
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• 2003

느타리 세균성갈색무늬병 병원균 P. tolaasii의 전염원을 파악하기 위하여 느타리 재배사에서 사용하고 있는 물로부터 PCR방법을 이용하여 병원세균을 검출한 결과, 주로 충북지방의 느타리 재배사에서 수집된 57개 물 시료 전체의 28.1%인 16개 물에는 $m{\ell}$당 1.000 cfu 이하의 일반 세균을 포함하고 있었으며 54.4%인 31개의 시료가 1,001-10,000 cfu, 10.5%인 6개의 시료가 10,001-100,000 cfu, 그리고 7%인 4개의 시료가 100,001 cfu 이상의 세균을 포함하고 있었다. P. tolaasii의 경우 nested-PCR로 검출하였을 경우 전체의 5.3%인 3곳의 물이 Immunocapture-nested-PCR로 검출하였을 경우 전체의 35.1%인 20개의 물 시료로부터 P. tolaasii 특이적 DNA가 증폭되었다. 물에 포함된 일반세균의 농도와 P. tolaasii 검출과는 상관없었다. 이상의 결과는 물로부터 병원균을 검출하는 방법으로 IC-nested-PCR 방법이 더 민감하고 우수한 방법이며 여러 곳의 느타리 재배사에서 사용하고 있는 물이 병원균 P. tolaasii으로 오염되어 있는 것을 제시하고 있다.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1110-1114
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• 2005

Six viruses of the genera Carlavirus (Garlic mosaic virus, GarMV, and Garlic latent virus, GarLV), Allexivirus (Garlic virus X, GarV-X, and Garlic mite-borne filamentous virus, GarMbFV) and Potyvirus (Leek yellow stripe virus, LYSV, and Onion yellow dwarf virus, OYDV) from Korean garlic plants with mosaic symptoms were simultaneously detected by multiplex RT-PCR and subsequently sequenced. An immunocapture RT-PCR for the detection of GarLV, LYSV, and OYDV was also performed. The coat protein phylogenetic analysis of the garlic viruses showed that the Korean isolates were most closely related to the isolates from China, Japan, Brazil, and Argentina. This study is the first report for the differentiation of six garlic viruses in Korea by simultaneous detection using multiplex RT-PCR.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1776-1781
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• 2012

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio-${\beta}$-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzymelinked immunosorbent assay and immunocapture RT-PCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

• Journal of Microbiology and Biotechnology
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• 제8권2호
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• pp.101-110
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• 1998

The extent of biological diversity being revealed by molecular techniques accentuates the need to develop methods to isolate and culture the large numbers of microorganisms that remain to be studied. The discovery and characterization of novel microorganisms will provide information useful in understanding microbial ecosystems and have the potential to lead to new products for the biotechnology industry. In this review, the use of innovative techniques and exploration of unusual ecosystems, that have begun to address the challenge of isolating the "uncultured" members of the microbial population, are examined.

• The Plant Pathology Journal
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• 제26권1호
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• 원예과학기술지
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• 제32권4호
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• pp.544-549
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• 2014

Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf and bulb samples showing characteristic symptoms of virus infection were collected in 2012, and 80 field samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The infection frequencies were 79% for LMoV, 5% for LSV, and 3% for CMV. The LMoV coat protein gene was amplified and cloned into the pET21d(+) expression vector to develop serological diagnostic tools to detect LMoV. The resulting carboxy-terminal His-tagged coat proteins were expressed in Escherichia coli strain BL21 (DE3) by induction with IPTG. The recombinant proteins were purified using Ni-NTA agarose beads and used as an antigen to produce polyclonal antibodies in laying hens. The resulting egg yolk immunoglobulin (IgY) specifically recognized LMoV from infected plant tissues in immunoblotting assays and had comparable sensitivity to that of a mammalian antibody. In addition, method of immunocapture RT-PCR using this IgY was developed for sensitive, efficient, and rapid detection of LMoV. Based on these results, large-scale bulb tests and detection of LMoV in epidemiological studies can be performed routinely using this IgY. This is the first report of production of a polyclonal IgY against a plant virus and its use for diagnosis.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5741-5745
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• 2013

Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. Materials and Methods: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. Results: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. Conclusions: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.