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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.
Israel Acute Paralysis Virus (IAPV)의 Nested PCR 검출법의 개발
김일욱(Il-Wook Kim),유미선(Mi-Sun Yoo),강민희(Min-Hee Kang),윤병수(Byoung-Su Yoon) 한국양봉학회 2009 韓國養蜂學會誌 Vol.24 No.2
We developed the nested PCR method for the detection of IAPV that was reported as a putative marker for colony collapse disorder (CCD). We analysed the sequence of IAPV (accession No.EF219380) to amplify IAPV gene. A pair of IAPV specific inner primers (IAPV-IF and IAPV-IR) was used for detection of 147 bp stable region of IAPV gene. The sensitivity test was performed with 164IAPV clone. The detection limit of direct PCR with IAPV-IF and IAPV-IR was 1pg. However, the detection limit of nested PCR with 2 pair of IAPV specific primers was calculated as 10fg. This results showed that the nested PCR method for the detection of IAPV were highly sensitive and applicable to the early detection and identification of IAPV.
Israel Acute Paralysis Virus (IAPV) 검출을 위한 PCR primer쌍들의 평가
김일욱(Il-Wook Kim),강민희(Min-Hee Kang),유미선(Mi-Sun Yoo),권순환(Soon-Hwan Kwon),윤충효(Choong Hyo Yun),윤병수(Byoung-Su Yoon) 한국양봉학회 2008 韓國養蜂學會誌 Vol.23 No.2
In order to evaluate primers designed to detect Israel acute paralysis virus (IAPV) that was reported as a putative marker for colony collapse disorder (CCD), cDNA which was synthesized from 10 honeybee suspected samples was examined by specific PCR with 9 different pairs of primers. In this study, specific amplicons were produced by PCR using AMIAPV, ORF2 and IAPVrDrp primer-pairs from IAPV-infected samples and also from Kashmir Bee Virus (KBV) infected samples. In case of PCR with IAPV162PF/ 113PR primer pairs, only IAPV-infected samples were detected with weak signal in some case. In addition, 4 pairs of primers were newly designed for the purpose of confirmational detection for IAPV. Using IAPV specific primers-pairs, named IAPV162PF/R, IAPV113PF/R, IAPV105SF/R and IAPV-gp2 F/R, only IAPV-infected samples were specifically detected.
조병옥,김일욱,문상흡 ( Byeong Ok Cho,Yil Wook Kim,Sang Heup Moon ) 한국화학공학회 1994 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.32 No.4
The correlations between the internal and the process variables in plasma etching were derived based on fundamental theories as well as experimental data from literatures. The rates of plasma etching in SF_6 system were calculated based on these correlations, which agreed with the reported experimental values within the same orders of magnitude. The etch rates calculated for various process conditions changed in the same trend as the experimentally measured ones. Reactive ion etching(RIE) in Cl₂/HBr/He system was performed under different pressures and electrical powers. Parameters in the correlation equations derived from literatures were replaced by three adjustable parameters, C₁, C₂, and C₃, which were determined by fitting the simulation profiles to the three experimental results. Trench contours simulated using these parameters agreed well with those obtained from the experiments.
Real-time PCR을 이용한 스트레스에 따른 벼의 Receptor like kinase (RLK) 유전자의 발현 변화 분석
강민희,김일욱,한상훈,윤충효,윤병수 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.4
In plant, Receptor-like kinases (RLKs) are protein family, though its function is not yet understood, consisted of a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. RLKs are involved in hormonal response pathways, cell differentiation, plant growth and development, self-incompatibility, and symbiont and pathogen recognition. In this study, expression levels of RLG1, RLG5, RLG6, RLG#6, RLG8, RLG10, RLG17, RLG18 and RLG20 were analyzed by Real-time PCR, when rice (Oryzae sativa) was treated abiotic stress. The expression levels of all RLGs were compared each other by analyzed value of threshold cycles (CT). Consequently, RLGs were suppressed by NaCl as salinity stress, and expression of each RLK genes were showed difference treated salicylic acid and wound, respectively. However, All RLGs were induced under low temperature condition. Therefore, our results indicate protection-function of RLK genes to be an early response of rice against cold weather.