• 한국육종학회지
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• 제43권4호
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• pp.265-272
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• 2011

This study is to generate SCARs markers for identification of Perilla species. A SCAR is a genomic DNA fragment at a single genetically defined locus that is identified by PCR amplification using a pair of specific oligonucleotide primers. We derived SCARs by sequencing and cloning the both ends of the amplified products of RAPD markers. Sixteen sequence-specific primers were synthesized from eight RAPD markers, which were completely sequenced. We developed the species-specific SCAR markers which could be used successfully in detecting genetic variation in four Perilla species. These markers could be used to verify species-origins of various forms of Perilla germplasms.

• The Plant Pathology Journal
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• 제15권5호
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• pp.287-294
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• 1999

A technique based on the polymerase chain reaction (PCR) for the specific detection of genus Phytophthora and Phytophthora cryptogea-P. drechsleri complex group was developed using nucleotide sequence information of ribosomal DNA (rDNA) regions. The internal transcribed spacers (ITS) including 5.8S were sequenced for P. cryptogea-P. drechsleri complex group and its related species. Two pairs of oligonucleotide primers were designed. Primer pair ITS1/Phy amplified ca. 240 bp fragment in 12 out of 13 specie of Phytophthora, but not in Pythium spp., Fusarium spp.and Rhizoctonia solani. Primer pair rPhy/Pcd amplified 549 bp fragment only in P. cryptogea-P. drechsleri complex group, but not in other Phytophthora spp.and other genera. Specific PCR amplification using the primers was successful in detecting Phytophthora and P. cryptogea-P. drechsleri complex group in diseased plants.

• Mycobiology
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• 제32권3호
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• pp.123-127
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• 2004

Sweet persimmons have been increasingly cultivated in the southern part of Korea. However, anthracnose disease caused by Colletotrichum species is one of the major hindrances in cultivation and productions. In this study, we used polymerase chain reaction(PCR) to detect Colletotrichum species with the AFLP(amplified fragment length polymorphism) method. In AFLP, we used E3(5'-GACTGCGTACCAATTCTA-3') and M1(5'-GATGAGTCCTGAGTAACAG-3') primer combination and, as a result, 262 bp segment was observed in Colletotrichum species only. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless sweet persimmon plants. Based on sequence data for specific segments, Co.B1(5'-GAGAGAGTAGAATTGCGCTG-3') and Co.B2(5'-CTACCATTCTTCTA GGTGGG-3') were designed to detect Colletotrichum species. The 220 bp segment was observed in Colletotrichum species only, but not in other fungal and bacterial isolates.

• Mycobiology
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• 제31권1호
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• pp.23-31
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• 2003

With universal primer ITS1-F, the specific DHJ2 primer was developed to detect the Ectomycorrhizal(ECM) root tips in soil and to identify the species of ECM fungi, as based on DNA sequences of rDNA stored in GeneBank of NCBI. This primer was designed with the common sites of rDNA of Amanita and Boletus, and was also designed with several DNA programs provided by NCBI. The DNA fragments synthesized by PCR were calculated to be 1,000 to 1,200 bps of DNA located to 18s to 28s rDNA to contain two variable sites of ITS, indicating much diversities for specific species or ecotypes of ECM fungi. The primer DHJ2 reacted with the genomic DNA's extracted from the tissues of basidiocarp at the rate of 73 of 80 fungi collected produced single bands with a 1,100 bps length. The DNA fragment synthesized with the genomic DNA that extracted from eight ECM tips of Pinus densiflora was confirmed and analysized to the rDNAs of ECM in full sequences, and informed to be a ECM fungal species in the forest.

• Fisheries and Aquatic Sciences
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• 제11권3호
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• pp.133-139
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• 2008

Molecular techniques, including restriction fragment length polymorphism(RFLP) and polymerase chain reaction-single strand conformational polymorph isms(PCR-SSCP), were developed to identify salted, dried threadfin(Eleutheronema tetradactylum) and white herring(Ilisha elongata) fish. Using PCR with universal primers, conserved 367-bp fragments of the cytochrome b gene were amplified from fresh fish samples and sequenced. The sequences were then searched for specific restriction sites. The digestion of the PCR products with the endonucleases AvaI, FokI, MboII, and MspI generated RFLP, which was used to identify the commercial products. Similarly, the amplified PCR-SSCP products were developed and the products tested. Overall, similar patterns were found in the majority of the fresh and processed products. Based on the results, both RFLP and PCR-SSCP were useful in determining and validating the authenticity of the fish species used to prepare the commercial salted, dried products. A similar approach can be applied to other species.

• 식물병연구
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• 제20권1호
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• pp.21-24
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• 2014

Plasmodiophora brassicae는 십자화과 작물에 뿌리혹병을 일으키는 주요 병원균이다. 본 연구에서는 뿌리혹병균의 신속 정확한 검출을 위해서 뿌리혹병균에 대한 새로운 종 특이적 프라이머를 개발하고자 하였다. 새롭게 개발된 프라이머들은 10종의 주요 토양병원균을 비롯하여 기주인 배추 DNA와는 반응하지 않고 P. brassicae와만 반응하는 특이성을 갖고 있었다. 그 가운데 Primer ITS1-1/1-2는 민감도 검정 결과, 10 spores/ml의 DNA까지 검출이 가능함으로써, first round PCR용임에도 불구하고 이전의 검출법 보다 감도가 높고 정확한 결과를 얻었다. Quantitative real-time PCR로 분석할 경우에는 더 적은 수의 포자까지 안정적으로 검출해 낼 수 있어 새로운 P. brassicae 종 특이적 프라이머로서의 유용성을 확인할 수 있었다.

• 식물병연구
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• 제17권3호
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• pp.369-375
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• 2011

Fusarium verticillioides(완전세대: Gibberella moniliformis)는 Gibberellea fujikuroi 종 복합체에 속하는 식물병원균으로서 옥수수의 줄기와 이삭에 썩음병을 일으킬 뿐 아니라 인축에 중독증을 일으키는 fumonisin 곰팡이 독소를 생산한다. 본 연구의 목적은 옥수수에 주로 발생하는 fumonisin 생성가능 G. fujikuori 종 복합체 소속 Fusarium 곰팡이 중 F. verticillioides와 그 외 F. proliferatum, F. fujikuori 등을 서로 구별할 수 있는 종 특이적 PCR primer 조합을 개발하는 것이다. RNA polymerase II beta subunit 유전자(RPB2)의 염기서열로부터 제작된 특이 primer 조합(RVERT1와 RVERT2)은 우리나라 옥수수에서 분리한 잠재적인 fumonisin 생성 G. fujikuori 종 복합체 균주 중 오직 F. verticillioides로부터 208 bp 크기의 단일 DNA 절편을 증폭하였다. 한편 F. verticillioides를 포함한 모든 조사균주는 fumonisin 생합성에 필수적인 FUM1 유전자를 포함하고 있었다. 개발된 특이 primer 조합의 검출한계는 분석 곰팡이 DNA 0.125 pg/${\mu}l$ 수준이었다. 한편, 같은 primer 조합으로 Fusarium spp.에 오염된 옥수수 시료의 게놈 DNA로부터 F. verticillioides 특이 DNA 절편이 증폭되었다. 이와 같은 결과를 종합할 때, 본 연구에서 개발된 primer 조합은 여러 곡물 시료에 오염되어 있는 F. verticillioides 균주의 검출과 종 동정에 유용하게 사용될 것이다.

• 미생물학회지
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• 제55권1호
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• pp.1-8
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• 2019

Due to the major role played by several species of Streptococcus in the etiology of periodontitis, it is important to assess the pattern of Streptococcus pathogenic pathways within the infected subgingival pockets using a bacterial specific 16S rRNA fragment. From the total of 50 patients with periodontitis included in the study, only 23 Streptococcal isolates were considered for further analyses, in which their 16S rRNA fragments were amplified and sequenced. Then, a comprehensive phylogenetic tree was constructed and in silico prediction was performed for the observed Streptococcal species. The phylogenetic analysis of the subgingival Streptococcal species revealed a high discrimination power of the 16S rRNA fragment to accurately identify three groups of Streptococcus on the species level, including S. salivarius (14 isolates), S. anginosus (5 isolates), and S. gordonii (4 isolates). The employment of state-of-art in silico tools indicated that each Streptococcal species group was characterized with particular transcription factors that bound exclusively with a different 16S rRNA-based secondary structure. In conclusion, the observed data of the present study provided in-depth insights into the mechanism of each Streptococcal species in its pathogenesis, which differ in each observed group, according to the differences in the 16S rRNA secondary structure it takes, and the consequent binding with its corresponding transcription factors. This study paves the way for further interventions of the in silico prediction, with the main conventional in vitro microbiota identification to present an interesting insight in terms of the gene expression pattern and the signaling pathway that each pathogenic species follows in the infected subgingival site.

• 대한약침학회지
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• 제12권1호
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• pp.13-20
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• 2009

Objective : This study developed species-specific PCR and PCR-RFLP to detect the adulteration of Fel ursi products with cattle and pig bile juices. Methods : All the primers for PCR and PCR-RFLP in this study were designed based on nucleotide sequences of cytochrome b genes in the mitochondria. Results : The species-specific PCR amplified a DNA fragment of 214, 214, 295, and 167 bp from Fel ursi product, bear fur, cattle bile juice, and pig bile juice, respectively. The survey using the speciesspecific PCR indicated that some of commercial Fel ursi products were adulterated with cattle and pig bile juices. PCR-RFLP using the restriction endonucleases, HaeIII and HinfI enabled differentiation among Fel ursi product, cattle bile juice, and pig bile juice. Bear furs from two animals showed variations in PCR-RFLP patterns with HaeIII. Discussion : The detection methods of the species-specific PCR and PCR-RFLP could be useful in eliminating adulterated Fel ursi products from the market.

• 생명과학회지
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• 제27권7호
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• pp.746-753
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• 2017

참조기는 민어과에 속하는 우리나라의 중요한 산업 어종 중 하나이다. 최근 과도한 남획과 해양 환경의 변화로 참조기의 어획량이 줄어들자 일부 유통과정에서 유사어종인 부세, 흑조기 및 긴가이석태를 참조기로 둔갑시키는 사례가 빈번하게 발생하고 있다. 이에 본 연구에서는 종 특이 primer를 사용하여 참조기, 부세, 흑조기 및 긴가이석태를 신속하게 분석할 수 있는 방법을 마련하였다. 약 1,400 bp의 미토콘드리아 COI 유전자 분석을 통하여 종간 특이성을 나타내는 단일염기다형성 유전자를 탐색하였으며, PCR 증폭산물의 크기를 고려하여 4개의 종특이적 정방향 primer를 제작하였다. 단일 PCR을 이용한 종간 교차반응을 통하여 최적의 PCR 조건을 확립하였으며, 이후 제작된 4개의 정방향 primer를 혼합하여 4종에 대한 다중 PCR 반응을 진행하였다. 증폭된 산물은 전기영동을 통해 크기에 따라 1,540 bp, 1,013 bp, 470 bp 그리고 182 bp로 분리되었으며, 각각 참조기, 흑조기, 부세, 긴가이석태로 명확하게 판별이 가능하였다. PCR 민감도 측정에서도 모든 종에서 $0.1ng/{\mu}l$의 농도까지 검출 가능함을 확인하였다. 따라서, 본 연구에서 개발된 참조기와 유사어종에 대한 종특이 다중 PCR 분석법은 정확도와 민감도가 우수하여, 불법 유통가능성이 있는 제품에 대한 신속하고 정확한 판별로 식품안전관리에 효과적으로 활용될 것이라 사료된다.