• Pediatric Infection and Vaccine
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• 제22권2호
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• pp.106-112
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• 2015

목적: 장기간의 항생제 치료는 중이염 어린이 환자의 중이액으로부터 원인균이 배양되는 것을 방해한다. 본 연구는 배양 음성 중이액으로부터 분자적 진단에 의한 신속한 균 검출 가능성 여부를 확인하고자 하였다. 방법: 폐구균 lytA 유전자를 표적으로 하는 PCR과 LAMP로 민감도와 특이도를 비교 결정하고, 임상중이액에서의 폐구균 검출에 적용하였다. 결과: PCR 기법에 의한 폐구균 검출 최소한계는 약 $10^4$ 집락형성단위(CFU)이고, LAMP의 검출 최소한계는 10 CFU에서 결정되었다. 한편 두 가지 검사법 모두 Haemophilus influenzae 와 Moraxella catarrhalis 에 대해 $10^6$ CFU 이상에서도 DNA를 증폭하지 않았다. 22개의 배양음성 중이액 중에서 12개 검체가 LAMP-양성(54.5%, 12/22)으로 확인되었고, 이들 12개 LAMP-양성 검체 중, 3개의 검체만이 PCR-양성으로 확인되었다(25%, 3/12). 본 연구의 결과는 LAMP 기법의 폐구균 검출 해상력이 PCR 기법에 비교하여 4배 이상 높음을 보여준다(P<0.01). 결론: lytA -특이 LAMP 기법은, 중이액 내의 타 병원균과는 교차반응 없이 10 CFU 폐구균의 DNA를 검출할 수 있는 고해상 기술로서, 중이액 폐구균 검출 및 폐구균 백신의 보급에 따른 백신 효과 평가에 적용이 기대된다.

• 한국식품위생안전성학회지
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• 제27권2호
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• pp.182-187
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• 2012

본 연구에서는 향신료 조제품인 고추다대기의 사용원료 확인을 위한 분자생물학적 기법을 이용한 시험법을 검토하였다. 시료 중 원료성분 확인을 위하여 고추, 마늘, 양파의 종 특이 프라이머를 이용하였으며, 대상 식품원료로는 고추다대기 6건을 선정하였다. 시료로부터 직접 유전자 추출 후 PCR을 실시하였으나 고추 등 사용원료의 확인이 어려웠다. 따라서 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR한 결과 6건 중 5건에서만 고추유전자 확인되었으며, 마늘 및 양파 성분은 모두 확인이 불가능하였다. 따라서 염 성분 제거 후 추출유전자의 증폭을 위하여 Whole Genome Amplification (WGA) 키트를 사용 후 PCR을 실시하였다. 그 결과 모든 시료에서 고추유전자(102 bp)를 확인하였으며, 양파를 함유하는 6건 및 마늘을 함유하는 4건에서 각각 양파(280 bp) 및 마늘(180 bp) 유전자를 확인하였다. 따라서 본 연구에서 검토된 고추다대기에 대한 시료 전처리법, 유전자추출법, 추출유전자증폭법 등은 고추다대기를 이용한 불량고춧가루 제조 시 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다.

• 식물병연구
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• 제17권1호
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• pp.66-74
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• 2011

2005년 울릉도에 자생하고 있는 산마늘에 대하여 바이러스병을 조사하기 위하여 성인봉 부근에서 61점의 시료를 채집하였다. 채집한 시료를 동정하기 위해 전자현미경 검경과 종 특이적 프라이머(GCLV, LYSV, SLV, OYDV) 및 속 특이적 프라이머(Allexivirus)를 이용하여 RT-PCR을 각각 수행하였다. 전자현미경 검경을 실시한 결과 61점의 시료중 4점의 시료에서 600~900 nm의 긴 사상형 입자가 관찰되었으며, RT-PCR 진단결과 SLV가 4점, 이 중 하나는 Allexivirus가 복합감염되었다. 바이러스에 감염된 산마늘은 외관상으로 병징을 나타내지 않았다. RT-PCR 분석결과 SLV와 Allexivirus로 동정된 울릉도 산마늘 바이러스의 외피단백질 유전자 염기서열을 비교 분석하였다. 분석결과, SLV로 분류된 울릉도 산마늘 바이러스 개체간은 약 99%의 상동성을 가지고 있었으며, 이전에 보고된 다양한 SLV와 GLV의 strain과의 염기 서열의 비교에서는 75.7~83.7%의 상동성을 보였으며 아미노산 서열의 비교는 89.2~97.0%의 높은 상동성을 나타냈다. 또한 산마늘에서 검출된 GarV-A는 이전에 마늘에서 보고된 GarV-A와 99.2% 외피단백질 유전자 염기서열 상동성을 보였으며, 아미노산 서열은 98% 상동성을 보였다. 그러나 다른 Allexiviruses(GarV-C, GarV-E, GarV-X, GMbMV and Shal X)와 아미노산 서열을 비교한 결과 60.6%~81.5%의 상대적으로 낮은 상동성을 보였다. 이러한 자료들을 바탕으로 산마늘에서 분리한 SLV와 GarV-A를 각각 SLV-Ulleungdo와 GarV-A-Ulleungdo로 명명하였다. 본 논문은 산마늘에서 발생하는 바이러스에 대한 첫 번째 보고이다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.147.1-147
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• 2003

Freesia, a member of the Iridaceae family, has fragant, tubular shaped flowers and is very popular ornamental plants in the world. Diseased freesia plants showing systemic leaf streak mosaic symptoms were collected from a cultivated farm in Kyonggi province, Korea in 2003, and its causal agents were investigated. Two viruses, Cucumber mosaic virus (Fr-CMV) and a potyvirus, were identified from the leaf tissues of the diseased freesia based on sequence analysis and host range tests. CMV-Fr could infect systemically on Chenopodium quinoa, C. amaranticolor, N. glutinosa, and N. benthamiana, and this biological property is distinguishable from ordinary strains of CMV. A filamentous potyvirus-shaped virus could not infect general indicator plants by mechanical inoculation. Single RT-PCR products was successfully amplified with a set of degenerate primers specific to the Potyvirus genus and total nucleic acids from the infected tissues, and was cloned into the pGEMT-Easy vector. Nucleotide sequences confirmed it belongs to the Potyvirus genus with either a new species or an isolate of Freesia mosaic virus (no information is available for the FrMV). This is the first report of FrMV in Korea and more characterizations of the two viruses are in progress.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1229-1239
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• 2005

Human immunodeficiency virus-like particles (HIVVLPs) with native conformations similar to that of the wild-type virion could be valid candidates for vaccine development. To this end, we used a Semliki Forest Virus (SFV) expression system to produce HIV- VLPs containing high quantities of native envelope proteins. Here, we described a single SFV replicon containing the HIV gagpol and env genes under the control of separate subgenomic promoters. Mature VLPs incorporating the Gag and Env proteins were detected in the supernatant of replicon-expressing cells by Western blot analysis. The HIV-VLPs showed the expected molecular density (1.14-1.18 g/ml) on a $20-60\%$ sucrose gradient; the particles were 100-120 nm in diameter and Env proteins were observed on their surfaces by immunogold electron microscopy. RT-PCR analysis of VLP-associated RNAs in mature HIV-VLPs revealed two SF V-derived RNA species (full-length and subgenomic). Immunization studies in Balb/c mice showed that these HIV-VLPs were capable of inducing both HIV-specific antibodies and cell-mediated immune responses. Taken together, our results indicate that the SFV replicon system is useful for the production of HIV-VLPs, which may be valuable candidates for an HIV vaccine.

• Journal of Ecology and Environment
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• 제29권2호
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• pp.143-149
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• 2006

This study was conducted to identify native ectomycorrhizal (ECM) fungi colonizing Pinus densiflora for revegetation of abandoned coal mines in Korea. Seedlings of P. densiflora growing on coal mining spoils of a study site in Samcheok were collected. ECM roots were observed under stereomicroscope and their DNA were extracted from each root tip for a seedling for molecular identification. A PCR primer pair specific to fungi, ITS1F and ITS4, was used to amplify fungal DNA. Restriction enzymes, Alul and Hinfl were used for restriction fragment length polymorphism (RFLP). Combined with RFLP profiles and sequence analysis, total twenty one taxa were identified from the ECM root tips. Basidiomycetous fungi including Thelephoraceae, Pezizales, Laccaria, Pisolithus and Ascomycetous fungi including ericoid mycorrhizal fungi were identified from this study. Results showed that the most frequently found in the study sites was a species in Thelephoraceae. A possible use of ECM fungi identified in this study for the revegetation of abandoned coal mines with P. densiflora was discussed.

• The Plant Pathology Journal
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• 제24권3호
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• pp.361-364
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• 2008

The complete nucleotide sequences of a Korean isolate of Potato virus X(PVX-Kr) has been determined. Full-length cDNA of PVX-Kr has been directly amplified by long template reverse transcription and polymerase chain reaction(RT-PCR) using virus specific 5'-end primer and 3'-end primer, and then constructed in a plasmid vector. Consecutive subclones of a full-length cDNA clone were constructed to identify whole genome sequence of the virus. Total nucleotide sequences of genome of PVX-Kr were 6,435 excluding one adenine at poly A tail, and genome organization was identical with that of typical PVX species. Comparison of whole genome sequence of PVX-Kr with those of European and South American isolates showed 95.4-96.8% and 77.4-77.9%, in nucleotide similarity, respectively. Sequenced PVX-Kr in this study and twelve isolates already reported could be divided into two subgroups in phylogeny based on their complete nucleotide sequences. Phylogenetic tree analysis demonstrated that PVX-Kr was clustered with European and Asian isolates(Taiwan, os, bs, Kr, S, X3, UK3, ROTH1, Tula) in the same subgroup and South American isolates(CP, CP2, CP4, HB) were clustered in the other subgroup.

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.294-300
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• 2009

Activation tagging that uses T-DNA vectors containing multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an activation tagged hairy root pool of ginseng (Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the biosynthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quantitative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of triterpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

• 한국어병학회지
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• 제16권1호
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• pp.51-59
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• 2003

Streptococcal infection is one of the most serious disease of cultured olive flounder, Paralychthys olivaceus in Korea and caused by more than one species. However, there has been considerable confusions about the taxonomic position of the fish pathogenic streptococci. In this study, We performed the randomly amplified polymorphic DNA(RAPD) pattern analysis to evaluate the possible classification in 8 streptococci isolated from diseased olive flounder and reference strains based on their DNA structure. RAPD PCR with DNA solution prepared by simple boiling and 10-mer random primer was appeared to be a good tool for discrimination of different streptococcal strains. Phenotypical characters by simple biological test and API 20 Strep corresponded well to the specific profiles of RAPD in streptococcal isolates of this study. Therefore, the RAPD profile was considered as one of differential characters to discriminate the streptococcal isolates from diseased olive flounder.

• BMB Reports
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• 제44권3호
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• pp.187-192
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• 2011

TGF-$\beta$ activated kinase-1 (TAK1) plays a pivotal role in developmental processes in many species. Previous research has mainly focused on the function of TAK1 in model organisms, and little is known about the function of TAK1 in hymenoptera insects. Here, we isolated and characterized the TAK1 gene from Apis cerana cerana. Promoter analysis of AccTAK1 revealed the presence of transcription factor binding sites related to early development. Real-time quantitative PCR and immunohistochemistry experiments revealed that AccTAK1 was expressed at high levels in fourth instar larvae, primarily in the abdomen, in the intestinal wall cells of the midgut and in the secretory cells of the salivary glands. In addition, AccTAK1 expression in fourth instar larvae could be dramatically induced by treatment with pesticides and organic solvents. These observations suggest that AccTAK1 may be involved in the regulation of early development in the larval salivary gland and midgut.