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Molecular Level Relationships of Purple Nonsulfur Bacteria and their Relatives
이상섭,윤병수,김재수,이현순,Lee, Sang-Seob,Yoon, Byoung-Su,Kim, Jae-Soo,Lee, Hyun-Soon The Microbiological Society of Korea 1994 미생물학회지 Vol.32 No.1
광합성 세균과 비광합성 세균에 속하는 종들 사이의 유연관계를 파악하기 위해 DNA 혼성화 방법을 실시하였다. 혼성화도는 종내 균주들 사이와 Rhodobacter capsulatus와 Rhodopseudomonas blastica 사이를(72-88%) 제외하고는 전체적으로 낮게 나타났다(2-35%). 광합성 세균 Rhodopseudommonas Palustris와 비광합성 세균 Pseudomonas aeruginosa ATCC 27853, Bradyrhizobium japonicu, 사이의 D%는 광합성 세균 사이의 D%보다 약간 높게 나타났다(26-33%). Rhodopseudomonas blastica와 Rhodobacter capsulatus 사이의 D%는 72%로 유전적 유연관계가 매우 높은 것으로 나타났다. DNA-DNA hybridization by kinetic method was carried out between species of purple nonsulfur photosynthetic bacteria and nonphotosynthetic bacteria. The degrees of homology percent were shown to be low (2-35 D%) with the exception of high homology % (72-88 D%) for strains within a species and between Rhodobacter capsulatus and Rhodopseudomonas blastica. The D% between the purple nonsulfur photosynthetic bacteria, Rhodopseudomonas palustris, and nonphotosynthetic bacteria, Pseudomonas aeruginosa ATCC 27853 or Bradyrhizobium japonicum were a little higher (26-33 D%) than the D% between any other photosynthetic bacteria. The homology % between Rhodopseudomonas blastica and Rhodobacter capsulatus was 72 D%, which showed genetic relationship.
역전사 실시간 Recombinase Polymerase Amplification (RT/RT RPA)에 의한 꿀벌 Black Queen Cell Virus의 신속 검출
임수진(Su Jin Lim),Giang Thi Huong Luong,민상현(Sang Hyun Min),왕지희(Ji Hee Wang),윤병수(Byoung-Su Yoon) 한국양봉학회 2016 韓國養蜂學會誌 Vol.31 No.1
Black Queen Cell Virus (BQCV) is one of pathogenic virus in honeybee which could be detected using reverse transcription real-time PCR (RT/RT-PCR). In this study, for rapid detection of BQCV, Recombinase Polymerase Amplification (RPA) was applied, and BQCV-specific reverse transcription real-time RPA (RT/RT-RPA) method was newly developed based on BQCV-specific RT/RT PCR. The Real-time RPA (RT-RPA) was performed at 37°C isothermal condition for 40 minutes. During the experiments, specific DNA amplifications were real-timely monitored using fluorescent detector. BQCV-specific DNA amplification could be detected from 3 min 26 sec after RPA reaction with specific DNA templates by RT-RPA, while 41 min 42 sec was required by qRTPCR with same quantities of initial templates. With generated cDNA from BQCV-infected honeybee, specific DNA amplification was recognized at 4 min 18 sec using RT-RPA, however, 66 min 5 sec was needed using Real-time PCR. Moreover, with reverse transcriptase and RPA solution, BQCVspecific DNA amplification could be detected at 8 min 36 sec from total RNA of BQCV-infected honeybee using one-step Reverse Transcription/Real-Time RPA (RT/RT-RPA).
꿀벌 6종 주요 병원체에 대한 초고속 다중 PCR 검출법의 개발
임수진(Su-Jin Lim),김정민(Jung-Min Kim),이칠우(Chil-Woo Lee),윤병수(Byoung-Su Yoon) 한국양봉학회 2017 韓國養蜂學會誌 Vol.32 No.1
PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than 103 molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.
꿀벌의 밀납 분비를 이용한 프라스틱 용기내 꿀벌집 생산기술
김동수(Dong-Su Kim),유미선(Mi-Sun Yoo),윤병수(Byoung-Su Yoon) 한국양봉학회 2008 韓國養蜂學會誌 Vol.23 No.4
Optimum conditions for production of honeycomb and honey in plastic jar using characteristics of beeswax excretion of honeybee were investigated. Based on our observations, application of comb-foundations in jar was more effective than simply beewax-treated jar in the benefits of time-reduction for comb-formation and honey-production. In this study, comb-foundations were placed in a large plastic cylinder form jars. Small central cylinderform comb-foundation was used with or without 16 plates (rectangles) of comb-foundations around small cylinder. In the experiments in field, the completion time of construction for honeycomb and honey stocks in comb were finished 11-31 days according to form of comb-foundation or numbers of jars per hive. Also, the productivity of comb-construction and honey in plastic jars were discussed based on experimental results and calculations.
축산물 잔류 sulfadimethoxine 검출용 ELISA kit 개발
김우택,김성희,윤병수,임윤규,Kim, Woo-taek,Kim, Seong-hee,Yoon, Byoung-su,Lim, Yoon-kyu 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.3
An enzyme linked immunosorbent assay (ELISA) was developed to screen residues of sulfadimethoxine (SDM) in edible animal products. An indirect competitive ELISA was allowed to compete with rabbit anti-SDM for binding to a limited amount of SDM-gelatin conjugate and SDM in serum samples. Sera was diluted 20 times with phosphate buffered saline (PBS) and boiled for 5 minutes to destruct immunoglobulins of serum. Detection limit of this competitive ELISA for SDM was 0.1 ppb or less. Among eight sulfonamide analogues tested for specifity, only sulfamonomethoxine showed significant cross-reaction in the assay. The EC-50 value for sulfamonomethoxine was 3.5 ppm. Recovery of SDM in spiked serum samples between 100 ppb and 500 ppb ranged from 110.7% to 128.9%.
초고속 이단계 PCR에 의한 Shiga 독소 타입 1의 신속 검출법
김일욱,강민희,권순환,조성학,윤병수,Kim, Il-Wook,Kang, Min-Hee,Kwon, Soon-Hwan,Cho, Seung-Hak,Yoon, Byoung-Su 한국미생물학회 2008 미생물학회지 Vol.44 No.3
Shiga 독소 생성 대장균(Shiga toxin-producing Escherichia coli; STEC)을 가장 빠르게 검출할 수 있는 초고속 이단계 PCR 방법을 개발하였다. 검색 대상 유전자는 STEC에서 생성되는 Shiga 독소(Shiga toxin; Stx)를 암호화하고 있는 유전자 stx1이며, 1쌍의 stx 유전자 특이 primer를 사용하여 검출을 수행하였다. 초고속 PCR (Ultra-rapid PCR)은 microchip 기반의 6 ${\mu}l$ PCR 용량의 Real-time PCR을 사용하고, PCR 회전의 각 단계 중 혼성과 중합을 한 단계로 하였을 뿐 아니라, 각 단계의 적용시간을 각 1초, 3초(해리, 혼성/중합)가 되게 극단적으로 줄여, 검사소요시간을 최소화하였다. 35회전의 PCR 진단에 사용된 시간은 6분38초였으며, 용융온도분석에서 stx1 특이 유전자가 검출되었음을 확인하는 데까지 총 7분 28초가 소요되었다. 또한 민감도 측정에서 $3{\times}10^0$ CFU/reaction까지 성공적으로 검출 가능함이 확인되었고, 용융온도분석에서 이 증폭산물은 일정한 $81.42{\pm}0.34^{\circ}C$의 용융온도를 갖는 것으로 확인되었다. 이 검사법을 다양한 STEC 균주들에게 적용하여 그 성능을 검증하였으며, 이로써 본 초고속 이단계 PCR 방법은 Shiga 독소 생성 대장균의 초신속 검출에 바로 적용될 수 있을 것으로 기대한다. Rapid detection-method for Shiga toxin type 1 that was produced from Shiga toxin-producing Escherichia coli (STEC) was developed by two-step ultra-rapid real-time (URRT) PCR. The specific primers were deduced from 80 bp stable region of stx type 1 (stxl) gene among various informations of STEC strains. URRT PCR is a microchip-based real-time PCR using 6 ${\mu}l$ of reaction volume with extremely short denaturation step and annealing/extension step (1 sec, 3 sec, respectively) in each cycle of PCR. Using the stx1-specific URRT PCR, 35 cycled PCR were finished in time of 6 min and 38 see, also measured 7 min and 28 see including melting temperature (Tm) analysis. The detection-limit of stxl-specific URRT-PCR was estimated until 3 colony forming units / PCR with products with stable Tm at $81.42{\pm}0.34^{\circ}C$. In the applications to various STEC strains and contaminated genomic DNAs, stx1-specific URRT-PCR were tested and shown that it would be expected an useful method for the rapid detection of stx1-coded STEC strains.