• The Plant Pathology Journal
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• 제24권2호
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• pp.159-163
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• 2008

Accurate identification of pine wood nematode, Bursaphelenchus xylophilus is a prerequisite to diagnose the pine wilt disease. However, a fungivorous nematode, B. mucronatus is highly similar to B. xylophilus and it is difficult to differentiate these two species by morphological features. A molecular diagnosis method, ITSRFLP was applied for the identification of B. xylophilus and B. mucronatus from Korea. Genomic DNA was extracted from a single individual nematode and ITS DNA was amplified by PCR. The size of PCR product was approximately 900bp and the sequence data were obtained after cloning. Amplified ITS was digested by 5 different restriction enzymes (Rsa I, Hae III, Msp I, Hinf I, and Alu I) and provided a discriminatory profile for B. xylophilus and B. mucronatus. Besides, B. mucro- natus was determined to have 2 different genotypes, East Asian type and European type also clearly separated by Rsa I and Hae III digestion. European type of B. mucronatus is recently collected from Pinus koraiensis and has not been reported before. ITS sequnce data were analyzed by Restriction Mapper program and the result supported ITS-RFLP pattern. These data indicated that PCRRFLP method is an accurate and simple way for identification of Bursaphelenchus species.

• ALGAE
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• 제32권3호
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• pp.189-197
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• 2017

To quantify the abundance of the harmful dinoflagellate Cochlodinium polykrikoides in natural seawaters, we developed the innovative procedure using a droplet digital PCR (ddPCR) with C. polykrikoides-specific primers targeting the internal transcription sequence (ITS). The abundance of C. polykrikoides was estimated by the specific copy number of target ITS DNA segments per cell in cultures and natural water samples. The copy number per C. polykrikoides cell as acquired by ddPCR was $157{\pm}16$, which was evaluated against known cell numbers through a simplified protocol preparing DNAs. The abundances of C. polykrikoides in the waters of different locations estimated by ddPCR agreed with the number of cells visually counted under a microscope. This protocol was used to measure the abundance of C. polykrikoides close to and further off the southern coast of Korea in August of 2016 and 2017. The practical application showed that this method can reduce time for analysis and increase accuracy.

• Journal of Plant Biotechnology
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• 제45권2호
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• pp.102-109
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• 2018

엉겅퀴는 일반적으로 이용되는 대표적인 다년생의 약용식물이다. 최근 국제적 추세에 따라 자국의 유전자원의 발굴, 보존 등이 강화됨에 따라 인접국가와 국내 자생 엉겅퀴 계통을 판별 할 수 있는 기준 설정에 관한 연구의 필요성이 대두되고 있지만, 분자생물학적 판별 기술의 개발은 아직 미흡한 실정이다. 본 연구에서는 국내 토종과 해외 유래 엉겅퀴종의 기원을 판별하기 위해 핵의 리보솜에 존재하는 ITS 유전자단편에서 SNP를 이용한 판별 프라이머를 확보하였으며, 이를 보완하여 보다 신속하게 판별하기 위하여 ARMS-PCR 및 HRM 기술을 이용한 판별 마커와 그 조건을 확립하였다. 또한, 국내 종 특이적 프라이머들을 이용한 정량적 PCR 분석방법을 이용해 두 가지 종의 genomic DNA의 혼합 여부를 판별하였다. 그러므로, 본 연구에서 개발된 SNP 마커는 다양한 지역 또는 국가에서 서식하는 엉겅퀴 종들의 신속한 확인을 위해 매우 유용하게 이용될 것으로 생각된다.

• 생명과학회지
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• 제20권4호
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• pp.578-583
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• 2010

세균 및 진균류의 증식을 억제하는 능력이 있는 것으로 보고된 누로와 대계의 추출물을 사용하여 Mycobacterium smegmatis 및 Mycobacterium fortuitum의 증식을 억제하는 능력이 있는지를 시험하였다. 그 결과, 누로의 추출물에서는 증식억제능을 발견할 수 없었으나, 대계의 추출물에서는 뚜렷한 증식억제능이 관찰되었다. 따라서 본 연구에 사용된 대계(엉겅퀴)에 대한 분류학적 또는 진화적 분석을 수행하기 위하여 genomic DNA를 추출한 후, ITS1, 5.8S rRNA 유전자 및 ITS2를 포함하는 부분을 PCR로 증폭시켰다. PCR 산물의 염기서열을 분석한 결과, 733-bp의 염기서열이 얻어졌고, 이것을 GenBank에 등록하였다(accession number GU188570). 이렇게 얻어진 염기서열을 사용하여 BLAST analysis를 수행한 결과, 염기서열이 일치하는 생물체는 아직까지 GenBank에 보고된 적이 없고, 가장 가까운 식물들로는 귀화식물로서 전국적으로 분포하는 Carduus crispus (지느러미엉겅퀴) 및 현재까지 국내에 자생하는 것으로 보고된 적이 없는 Carduus defloratus로서 각각 3개씩의 염기가 다른 것으로 나타났다.

• 한국균학회지
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• 제40권2호
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• pp.78-85
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• 2012

20 mer의 URP primers 가 한국 재래적미의 반복배열 DNA 염기서열로부터 고안되어 다양한 곰팡이 종의 PCR 다형성검출에 적용 되어 왔다. URP-PCR 방법은 PCR반응중 $55^{\circ}C$이상의 고온에서 annealing반응을 함으로 하여 재생적인 PCR결과를 얻을 수 있으며 효모균류에서 담자균류의 고등 균류까지 33속, 142 종, 1,489 균주의 다양한 곰팡이 종의 유전체 DNA에 적용되어 그 유용성이 평가 되어 왔다. 본 논문은 URP-PCR의 특성과 지금까지 다양한 곰팡이 종 다양성 검정결과를 종합하여 보고 한다.

• ALGAE
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• 제37권4호
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• pp.281-291
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• 2022

Quantifying the abundance of Chattonella species is necessary to effectively manage the threats from ichthyotoxic raphidophytes, which can cause large-scale mortality of aquacultured fish in temperate waters. The identification and cell counting of Chattonella species have been conducted primarily on living cells without fixation by light microscopy because routine fixatives do not retain their morphological features. Species belonging to the Chattonella marina complex, including C. marina and C. marina var. ovata, had high genetic similarities and the lack of clear morphological delimitations between the species. To estimate the abundance of C. marina complex in marine plankton samples, we developed a protocol based on the droplet digital polymerase chain reaction (ddPCR) assay, with C. marina complex-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA. Cell abundance of the C. marina complex can be determined using the ITS copy number per cell, ranging from 25 ± 1 for C. marina to 112 ± 7 for C. marina var. ovata. There were no significant differences in ITS copies estimated by the ddPCR assay between environmental DNA samples from various localities spiked with the same number of cells of culture strains. This approach can be employed to improve the monitoring efficiency of various marine protists and to support the implementation of management for harmful algal blooms, which are difficult to analyze using microscopy alone.

• 대한본초학회지
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• 제29권3호
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• pp.19-26
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• 2014

Objectives : Aucklandiae Radix (Muxiang) one of important herbal medicines in oriental medicine, is defined as the dried root of Aucklandia lappa (Asteraceae). Owing to the similarities in the morphology and name, Inulae Radix (Tu-Muxiang) and Vladimiriae Radix (Chuan-Muxiang) as well as Aristolochiae Radix (Qing-Muxiang) originated from other medicinal plants are often used as substitutes and/or adulterants of Aucklandiae Radix. Therefore, a reliable authentication of these herbal medicines is necessarily for the public health and prevention of misuse. Methods : 32 samples of medicinal plants supplying Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix were collected in Korea and China. The ITS (Internal transcribed spacer) nucleotide sequences of samples were determined. The PCR primers to amply DNA marker of each herbal medicine were designed basing on the specific ITS regions showing differences in the sequences among medicinal plants. Results : Primer set Al R/IS F designed in this work amplified 220 bp PCR product only in samples of Aucklandiae Radix. In contrast, primer set Ih F/IS R, Vs R/IS F, and AcR F1/Ac R amplified 250 bp product, 356 bp prouct, and 516 bp product respectively to identify Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. Conclusions : The primers designed basing on the nucleotide sequences of ITS regions appearing differenced in the sequences among medicinal plants amplified the DNA markers for the identification of Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. These herbal medicines were more efficiently identified by multiplex PCR method using all primers in a single PCR process.

• 산업기술연구
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• 제28권A호
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1928-1935
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• 2015

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (μ_max and K_s). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.68-76
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• 2006

Despites many attempts to explore the microbial diversity in kimchi fermentation, the predominant flora remains controversial to date. In the present study, major lactic acid bacteria (LAB) were investigated in Chinese cabbage kimchi in the early phase of fermention. For the samples over pH 4.0, viable cell counts of Leuconostoc and Pediococcus were $10^6\;cfu/ml$ and below $10^2\;cfu/ml$, respectively, and 20 isolates out of 172 were subjected to a biochemical identification (API 50 CH kit) as well as molecular-typing methods including ITSPCR with a RsaI digestion and 16s rRNA gene sequence analysis for species confirmation. Seven isolates were nicely assigned to Lb. brevis, 6 to Leuconostoc spp. (2 mesenteroides, 2 citreum, I carnosum, I gasicomitatum), 4 to Weissella (3 kimchii/cibaria, 1 hanii) and 2 to other Lactobacillus spp. (1 farciminis, 1 plantarum). On the other hand, the biochemical identification data revealed 9 strains of Lb. brevis, 6 strains of Leuconostocs,2 strains of Lb. plantarum and 1 strain each of Lb. coprophilus and Lactococcus lactis. However, a single isolates, YSM 16, was not matched to the ITS-PCR database constructed in the present study. Two Lb. brevis strains by API 50 CH kit were reassigned to W kimchii/cibaria, Lb. coprophilus or W hanii, respectively, judging from the results by the above molecular typing approaches. As a whole, the identification data obtained by the biochemical test were different from those of ITS-PCR molecular method by about $63\%$ at genus-level and $42\%$ at species-level. The data by the ITS-PCR method conclusively suggest that predominant LAB species is probably heterolactic Lb. brevis, followed by W kimchii/cibaria, Leuc. mesenteroides, and Leuc. citreum, in contrast to the previous reports [3] that Leuc. mesenteroides is the only a predominant species in the early phase kimchi fermentation.