• The Plant Pathology Journal
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• 제26권4호
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• pp.409-416
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• 2010

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

• 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.

• Fisheries and Aquatic Sciences
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• 제7권3호
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• pp.122-129
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• 2004

Harmful Cochlodinium polykrikoides is a notorious harmful algal bloom (HAB) species that is causing mass mortality of farmed fish along the Korean coast with increasing frequency. We analyzed the sequence of the large subunit (LSD) rDNA D1-D3 region of C. polykrikoides and conducted phylogenetic analyses using Bayesian inference of phylogeny and the maximum likelihood method. The molecular phylogeny showed that C. polykrikoides had the genetic relationship to Amphidinium and Gymnodinium species supported only by the relatively high posterior probabilities of Bayesian inference. Based on the LSU rDNA sequence data of diverse dinoflagellate taxa, we designed the C. polykrikoides-specific PCR primer set, CPOLY01 and CPOLY02 and developed PCR detection assays for its sensitive, accurate HAB monitoring. CPOLY01 and CPOLY02 specifically amplified C. polykrikoides and did not cross-react with any dinoflagellates tested in this study or environmental water samples. The effective annealing temperature $(T_{p})$ of CPOLY01 and CPOLY02 was $67^{\circ}C$. At this temperature, the conventional and nested PCR assays were sensitive over a wide range of C. polykrikoides cell numbers with detection limits of 0.05 and 0.0001 cells/reaction, respectively.

• 미생물학회지
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• 제35권2호
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• pp.107-114
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• 1999

Fusarium 균에 속하는 16종 21균주를 대상으로 RAPD-PCR 방법을 이용하여 DNA 다형성을 분석하여 계통 유전학적 유연관계를 검토하였다. 40개의 random primer 로 시험하여 실험한 모든 종에서 다형성을 나타내는 11개의 primer를 선별하였다. RAPD 분석결과 평균 23.9개씩 모두 263개의 크기가 다른 RAPD 밴드들을 조사할 수 있었다. 각 primer에 대해 각각 독특한 DNA 다형성을 나타내었고, 증폭된 DNA 크기는 0.1-3.0 kb 범위에서 형성되었다. 각 균주간의 genetic similarity를 계산하여 유연관계를 dendrogram 으로 나타내었다. Genetic similarity 0.627을 기준으로 하여 크게 4그룹으로 나눌 수 있었다.

• 대한의생명과학회지
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• 제25권4호
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• pp.331-338
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• 2019

Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

• 한국해양학회지:바다
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• 제15권1호
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• pp.36-40
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• 2010

선박평형 수는 유독 와편모조류 및 다양한 미세조류의 국제적인 이동경로로 알려져 있다. 본 연구에서는 선박평형 수에 있는 와편모조류의 다양성을 조사하기 위하여 와편모조류 특이적인 PCR primer와 종 특이적인 real-time PCR 유전자 탐침자를 이용하였다. 선박평형 수 시료에 대한 광학현미경 조사에서는 와편모조류가 매우 낮은 농도로 관찰되었지만, SSU rDNA의 cloning 및 염기서열 분석 결과에서는 기생 와편모조류, 초미세플랑크톤, 어패류 폐사 원인종 등 다양한 종류가 확인되었다. 본 연구 결과는 종 톡이적 PCR primer와 같은 분자생물학적 방법이 선박 평형 수에 외래 유입종의 신속 정확한 진단에 유용함을 보여주고 있다.

• 한국자원식물학회지
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• 제19권2호
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• pp.336-339
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• 2006

홍화 수집종의 RAPD 분석을 통한 유전적 다양성 및 유연관계를 밝히고 품종군을 분류하여 품종육성의 기초자료로 활용코자 시험한 결과는 아래와 같다. RAPD 분석에 적용한 37개의 Primer 중 25개의 적정 primer를 선발하였고, 증폭원 PCR산물은 $0.1{\sim}4.0kb$에서 재현성있는 band를 보였으며 각 primer에 의해 증폭된 band의 수는 $1{\sim}9$개로 다양하였고 평균 4.8개였다. PCR 반응에 사용된 25개의 primer에서 120개의 band가 관찰되었으며 다형성을 보이는 band될 수는 23개(19.2%)였다. RAPD-PCR에 의해 얻어진 dendrogram에서 유연계수 0.042를 기준으로 합을 했을 때 6개 군으로 분류되었고 II군과 III군은 각각 12종(38%)씩 속하였다.

• 한국잠사곤충학회지
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• 제38권1호
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• pp.7-12
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• 1996

누에 RAPD-PCR 최적조건에 대해 실험한 결과, 중합효소 연쇄반응(PCR)의 최적조건은 반응액 25$\mu$l에 대해 주형 DNA 30ng, dNTP mixture 200$\mu$M, primer 300mM, Taq DNA Polymerase 1.0unit 및 Mg2+ 1.5mM로 판단되었으며, 또 상기의 실험조건을 기본으로 하여 annealing 온도를 탐색한 결과 35$^{\circ}C$-42$^{\circ}C$에서 DNA의 증폭이 안정적임이 밝혀졌다. 또한 동일한 품종내에서 개체간 및 암수간의 DNA 다형성은 인정되지 않았고, 품종간 DNA 다형성은 OPM 04 primer를 사용한 결과 5개의 RAPD 마커로 인정 되었다. 양친간 다형성이 인정된 primer를 사용하여 F2 33개체에 대해 DNA를 증폭시킨 결과 800 bp band가 3 :1 로 분리되었으므로 누에 품종간 유전적 유연관계 및 유전자 지도작성의 가능성을 제시했다.

• The Plant Pathology Journal
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• 제34권2호
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• pp.104-112
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• 2018

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss's bacterial wilt and blight of maize, Stewart's wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was $2pg/{\mu}l$ of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to $8.8{\times}10^3cfu$ to $7.84{\times}10^4cfu$ per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

• The Plant Pathology Journal
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• 제32권1호
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• pp.53-57
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• 2016

Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B.cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.