• International Journal of Oral Biology
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• 제46권4호
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• pp.155-159
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• 2021

This study aimed to develop strain-specific polymerase chain reaction (PCR) primers to detect Fusobacterium hwasookii KCOM 1249^T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1256, F. hwasookii KCOM 1258, and F. hwasookii KCOM 1268 on the basis of nucleotide sequences of a gene specific to each strain. The unique genes for each F. hwasookii strain were determined on the basis of their genome sequences using Roary. The strain-specific PCR primers based on each strain-specific gene were designed using PrimerSelect. The specificity of each PCR primer was determined using the genomic DNA of the 5 F. hwasookii strains and 25 strains of oral bacterial species. The detection limit and sensitivity of each strain-specific PCR primer pair were determined using the genomic DNA of each target strain. The results showed that the strain-specific PCR primers correspond to F. hwasookii KCOM 1249^T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1258, F. hwasookii KCOM 1256/F. nucleatum subsp. polymorphum KCOM 1260, or F. hwasookii KCOM 1268/Fusobacterium sp. oral taxon 203 were developed. The detection limits of these strain-specific PCR primers ranged from 0.2 to 2 ng of genomic DNA for each target strain. The results suggest that these strain-specific PCR primers are valuable in quality control for detecting specific F. hwasookii strains.

• Archives of Pharmacal Research
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• 제26권8호
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• pp.601-605
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• 2003

Traditional taxonomic methods used for the identification and differentiation of ginsengs rely primarily on morphological observations or physiochemical methods, which cannot be used efficiently when only powdered forms or shredded material is available. Randomly amplified polymorphic DNA (RAPD) was used to determine the unique DNA profiles that are characteristic not only of the genus Panax but also of various Panax subgroups collected from five different countries. RAPD results of OP-5A primer showed a specific single band that is characteristic of all ginseng samples. RAPD results of OP-13B primer demonstrated that OP-13B primer could be used as a unique RAPD marker to differentiate Panax species. These results support that this approach could be applied to distinguish Korean Ginseng (Panax ginseng) from others at the molecular level.

• 한국균학회지
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• 제27권5호
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• pp.337-340
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• 1999

rDNA내의 ITS region의 염기서열 분석 결과를 토대로 19종의 Phellinus 속균중 Phellinus linteus를 특이적으로 동정할 수 있는 primer를 제작하였다. 이 특이 primer는 ITS1과 ITS2내에 위치하며 이들 spacer region에 인접해 있는 universal Primer내에 위치해 있다. 총 4개의 Primer(universal primer인 ITS-1F와 ITS-4 그리고 특이 primer인 PL-F와 PL-R)가 한국에서 채집된 Phellinus 속균중 Phellinus linteus를 동정하는데 사용되었다. Phellinus linteus의 증폭된 DNA크기는 800bp(ITS-1F/ITS-4)와 720 bp(ITS-1F/PL-R과 PL-F/ITS-4)에 해당하는 2개의 band, 그리고 610 bp(PL-F/PL-R)인 것으로 나타났다. 한국에서 채집된 23종의 Phellinus속균중 13종이 Phellinus linteus인 것으로 확인되었다.

• Mycobiology
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• 제30권4호
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• pp.197-201
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• 2002

Specific primer sets based on ribosomal DNA(rDNA) internal transcribed specer(ITS) sequences were designed for rapid detection of Phellinus linteus and P. baumii. Polymerase chain reaction(PCR) with these primers produced unique bands for each Phellinus species. The annealing temperature range is from $40^{\circ}C\;to\;55^{\circ}C$. The length of PCR products(P. linteus and P. baumii) using designed combinative primer sets of PL1F, PL2R, PB1F, PB2R, ITS5F and ITS4R, were from 520 by to 730 bp. Fifteen strains of Phellinus species including P. linteus, P. baumii, P. weirianus, P. johnsonianus, P. rhabarberinus, P. pini, P. gilvus, P. igniarius, P. nigricans and P. laevigatus were examined in this study. Five strains, including two isolated strains of P. linteus(MPNU 7001 and MPNU 7002), and two isolated strains of P. baumii(MPNU 7004 and MPNU 7005) were shown to have about 520 bp (PL1F-PL2R), 700 bp (TTS5F-PL2R) and 600 bp (PB1F-ITS4R) -sized PCR single bands respectively. This molecular genetic technique provided a useful method for rapid detection and identification of P. linteus and P. baumii.

• Journal of Animal Science and Technology
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• 제48권5호
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• pp.637-650
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• 2006

한국의 예산과 당진에서 각각 채취된 붕어 (Carassius auratus)와 떡붕어 (Carassius cuvieri)로부터 genomic DNA를 분리 추출하여 반복해서 PCR로 증폭시켰다. 선택된 7개의 RAPD primer를 이용하여 primer 당 total loci, shared loci by each species, polymorphic 및 specific loci를 얻어냈다. 2종의 붕어로부터 primer와 2지역간에 banding patterns의 복잡성이 두드러지게 나타났다. DNA fragment의 분자적 크기는 150bp에서부터 1,600bp까지 커다란 차이를 나타내었다. 본 연구에서 CCY 붕어 종에서는 458개의 loci가 나타났고, CCD 떡붕어 종에서는 358개의 loci가 확인되었다. 또한 CCY 붕어 종에서는 84개의 polymorphic loci (18.3%)가 확인되었고, CCD떡붕어 종에서는 48개의 polymorphic loci (13.4%)가 확인되었다. CCY 붕어 종에서는 154개의 shared loci가 나타났으며, 이는 primer당 평균적으로 22개의 loci로 확인되었다. 또한 CCD떡붕어 종에서는 187개의 shared loci가 확인되었고, 평균해서 primer 당 26.7개의 loci가 나타났다. CCY붕어 종과 CCD 떡붕어 종의 polymorphic loci는 각각 84개와 48개로 확인되었다. 모든 붕어와 떡붕어 시료의 평균적인 BS value를 기초로 해서 CCY 붕어 종의 similarity matrix를 조사해 본 결과 0.434로부터 0.868까지 나타났고, CCD 떡붕어 종의 값은 0.449로부터 0.924까지 확인되었다. CCY 붕어 종내의 평균적인 BS value는 0.641±0.013이고, CCD 떡붕어 종내의 BS value의 평균값은 0.684±0.013을 나타내었다. 결과적으로 CCD 떡붕어 종내의 개체의 BS value 평균값이 CCY 붕어 종내의 평균값보다 높게 나타났다. 2 붕어와 떡붕어간의 평균적인 BS value은 0.484±0.007 (0.307～0.682)를 나타내었다. 7개의 primer를 사용하여 얻어진 dendrogram은 cluster 1 (AURATUS no. 01～AURATUS no. 11), cluster 2 (CUVIERI no. 12～CUVIERI no. 21) 및cluster 3 (CUVIERI no. 22)와 같이 3개의 유전적 클러스터로 나뉘어졌다. CCY 붕어 종내의 8번째 개체 (AURATUS no. 08)와 9번째 개체 (AURATUS no. 09) 사이가 가장 가까운 유전적 관계 (0.064)를 나타내었다. 또한 CCY붕어 종의 11번째(AURATUS no. 11)와 CCD떡붕어 종의 17번째 (CUVIERI no. 17) 사이가 가장 먼 유전적 거리 (0.477)를 나타내었다. 결과적으로 볼 때 한국 및 대서양산 lobster (0.612), 갈치 (0.708), 동자개(0.714)에 비해서 상대적으로 낮은 유전적 거리를 나타내었다.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.91-95
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• 2004

원유로 오염된 토양의 생물학적 복원과정 동안 접종된 Nocardia sp. Hl7-1 균주를 확인하기 위해 165 rDNA sequence에 기초하여 균주에 특이적인 primer를 제작하였다 14균주의 16S rDNA sequence비교를 통해 제작된 4개의 primer set는 Hl7-1 균주를 특이적으로 검출할 수 있었다. 특히 NH169F-NH972R과 NH575F-NH972R의 primer set는 50 fg의 DNA와 $1.2${\times}$10^4$ cfu/g-soil의 균체농도까지 민감하게 검출할 수 있었다. 이 두 primer set는 원유로 오염된 토양의 bioremediation과정 동안 접종된 Hl7-1 균주의 특이적 검출을 가능케 하였으며, 이는 사용된 primer set에 의해 증폭된 PCR산물을 제한효소(EcoRI)로 절단한 결과와 DGGE를 통한 Hl7-1 균주의 확인을 통해 본 연구에서 제작된 primer set의 특이성을 검증하였다.

• 한국양식학회지
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• 제19권2호
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• pp.67-76
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• 2006

Genomic DNA isolated from two Porphyra species, P. tenera and P. dentate from Wando located on the southern coast of Korean peninsula was amplified by PCR reaction. The amplified products were separated by agarose gel electrophoresis (AGE) with decamer primer and stained with ethidium bromide. The eight arbitrarily selected primers OPA-04, OPA-06, OPB-01, OPB-08, OPB-10, OPB-11, OPB-14 and OPC-10 generated the shared loci, polymorphic, and specific loci. The size of DNA bands varies from 100 bp to 2,200 bp. The complexity of the banding patterns varies dramatically between the primers and two Porphyra species. A total of 528 loci observed were identified in P. tenera and 443 in P. dentata: 22 polymorphic loci (4.2%) in P. tenera and 30 (6.8%) in P. dentata. 154 shared loci observed, the average 19.3 per primer, were identified in P. tenera and 143 loci, the aver-age 17.9 per primer, in P. dentata species. The number of specific loci in P. tenera and P. dentata was 73 and 77, respectively. The average bandsharing value was $0.623{\pm}0.008$ with P. tenera and $0.560{\pm}0.009$ within P. dentata. The average bandsharing value between two Porphyra species was $0.408{\pm}0.004$, ranged from 0.305 to 0.564. The dendrogram obtained by the eight primers indicates four genetic clusters. The genetic distance between two Porphyra species ranged from 0.076 to 0.627. The individual no. 02 of P. tenera was genetically closely related to no. 01 of P. tenera(genetic distance=0.082). Especially, two entities between the individual DENTATA no.21 and DENTATA no. 19 of P. dentata showed the longest genetic distance (0.627) in comparison with other individuals used. In this study, RAPD-PCR analysis has revealed the significant genetic distance between two Porphyra species pairs (P<0.001).

• 한국균학회지
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• 제31권3호
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• pp.121-128
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• 2003

국내 유통균주에 대한 Phellinus속의 분류체계를 확립하고 종간구별을 위한 신속동정법을 개발하기 위해 계통학적 정보를 지니고 있는 ITS1, 5.8S rRNA및 ITS2부위의 염기서열을 밝히고 ITS1과 ITS2 부위의 다양한 염기서열을 이용하여 종 특이적인 유전자 탐침을 개발하였다. 본 연구에서 조사된 바에 의하면, 상황버섯으로 시판되고 있는 국내유통균주는 총 9균주로서 P. pini가 4종, P. baumii가 3종, P. igniarius와 P. linteus가 각각 1종으로 확인되었으며 P. gilvus은 조사되지 알았다. 그 중 P. pini는 대부분 중국에서 수입된 제품이었고 P. igniarius를 포함한 P. baumii는 주로 북한산 제품으로 조사되었다. 한편 P. linteus와 P. baumii는 변이가 높은 ITS1 , ITS2 부위에서도 다른 종에 비하여 높은 상동성을 나타내어 종 특이적인 유전자 탐침이 유효하지 않은 반면에 P. igniarius, P. pini, P. gilvus종에 대한 detection primer로 각각 IF1-IR3, PF1-PF3, GF2-FR4 primer는 유전자 탐침이 유효함을 확인하였다. 유연관계가 아주 가까운 P. linteus와 P. baumii에 대한 정확한 분석을 위해서는 변이가 심한 ITS 부위와 보존성이 높은 18S rDNA 부위의 염기서열을 함께 분석, 비교하여야 가능하리라 사료된다.

• 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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• 제29권1호
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• pp.99-104
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• 2013

To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was $51.9^{\circ}C$. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.