• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권6호
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• pp.482-489
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• 2004

Bacillus species were observed and quantified by molecular approaches, using the 16S rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth of Bacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations by in situ hybridization, using Bacillus-specific probes, indicated that Bacillus strains were inhabited in the plant and their num­bers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using a Bacillus-specific primer set, and the amount of DNA originating from various Bacillus species was maximally $1.91%\$ of the total DNA in the wastewater treatment tank. Clone library analysis using the Bacillus-specific primers suggested that, while the population was no­ticeably increased, the phylogenetic diversity of the increasing Bacillus species was very low.

• Asian-Australasian Journal of Animal Sciences
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• 제16권3호
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• pp.425-429
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• 2003

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis and could be a causative agent of food poisoning, it produces various superantigenic exotoxins which have a great public health significance. A total of 72 S. aureus clinical isolates from dairy farms located in Kyunggi Province Korea were examined for the species identification by biochemical method, and for the detection of $\beta$-lactamase, enterotoxin and other exotoxins genes by PCR. The results of species identification by biochemical method agreed with those of PCR done with species specific primer STA-AU. $\beta$-lactamase is an enzyme closely associated with the resistance to antibiotic penicillin, which is an important means of treatment of mastitis, all the isolates were positive for the presence of genes encoding $\beta$-lactamase, which were reproduced in penicillin susceptibility disc assay. Six types of toxin genes, Staphylococcal enterotoxin (SE)A, SEB, SEC, SEE, toxic shock syndrome toxin (TSST-1) and exfoliative toxin A (ET A) were detected in 72 isolates by PCR associated genotypic method in this study, none of the isolates carried the genes for enterotoxin D (SED) and exfoliative toxin B (ETB). The occurrence rate of exotoxin genes rated as 12.5%, and the precision of the PCR identification results has been confirmed using the reference strains.

• Food Science and Biotechnology
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• 제18권5호
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Journal of Crop Science and Biotechnology
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• 제10권1호
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• pp.27-32
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• 2007

RAPD, Inter-SSR, and AFLP markers were used to assess the genetic diversity of lettuce cultivars and the phylogenetic relationships in Lactuca spp. A total of 216 polymorphic bands from seven RAPD primers, four Inter-SSR primers, and five AFLP primer combinations were used to elucidate the genetic similarity among lettuce cultivars. Forty-four lettuce accessions were subdivided into discrete branches according to plant type: crisphead, butterhead, and stem type, with some exceptions. The leafy- and cos-type accessions were intermingled in other groups with no discrete branch indicating that these are more diverse than others. Three accessions, including the Korean cultivar 'Cheongchima', the Korean local landrace 'Jinjam', and the German cultivar 'Lolla Rossa' were classified as the most diverse accessions. Twenty bands were unique in specific cultivars. Among these, three were specific in a plant type; one in Korean leafy type, one in crisphead type, and one in cos type lettuce. In the phylogenetic analysis among Lactuca species, L. saligna, L. serriola, and L. georgica clustered in a sister branch of the L. sativa complex. Two L. virosa accessions show the highest intra-specific relationships. L. perennis outlied from all the other Lactuca species at a genetic similarity of 0.53 and clustered with two Cichorium species, C. intybus and C. endivia, with genetic similarity of 0.67. The phylogenetic tree was supported by data from polymorphism of chloroplast genome which was revealed by PCR-RFLP.

• 한국균학회지
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• 제46권2호
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• pp.186-192
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• 2018

PCR 기술을 이용하여 고추와 토마토의 탄저병을 일으키는 Colletotrichum coccodes 균을 빠르고 정확하게 검출하는 방법을 개발하였다. Colletotrichum 13종 22개 균주로 부터 translation elongation factor 1 alpha 유전자를 분석하여 C. coccodes에 특이적인 coccoTef-F/cocco Tef-R primer set를 제작하였다. 제작된 primer를 사용한 결과 일반 PCR 방법으로 10 ng, real-time PCR 방법으로는 10 pg 수준에서 C. coccodes가 특이적으로 검출이 가능하였다. 인공적으로 C. coccodes에 감염시킨 고추와 토마토 종자에서도 일반 PCR 방법과 real-time PCR 방법 모두 C. coccodes검출이 가능하였다. 본 연구에서 개발한 PCR 방법은 수출입되는 종자에서 신속하고 정확하게 탄저병균 C. coccodes를 특이적으로 검출하는데 활용될 수 있을 것이다.

• 한국균학회지
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• 제26권4호통권87호
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• pp.574-582
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• 1998

1996년부터 속리산 법주사 주변과 그 외의 지역에서 Pholiota species 5종을 채집하여 Pholiota adiposa와 Pholiota species로 동정하였다. 이 버섯들을 다른 버섯들과 random primer에 의한 RAPD를 실시하여 형태적 분류와 연관시켜 본 결과, primer #28(1.5kb와 1.0kb 사이)에서 Pholiota adiposa와 Pholiota sp.의 공통밴드가 형성되었고, primer #36(750과 500bp 사이)와 OPD-18(760 bp)에서는 검은비늘버섯만의 특이적 밴드가 나타났다. 모균주와 이를 접종원으로 하여 수확한 자실체의 DNA에서는 동일한 모양의 다형적 밴드가 형성되었다. 또한 채집된 5종의 검은비늘버섯은 약간씩 다른 밴드적 차이를 나타내었다. 검은비늘버섯의 포자지문법을 통한 단포자분리에 의한 mating 결과 tetrapolar형을 관찰하였는데, 이는 Arita와 Mimura(1969)가 이미 P. adiposa와 P. nameko가 bipolar 형이라고 한 것과 다른 결과이다. 본 실험은 PCR-RAPD를 이용하여 하나의 버섯에서 얻은 담자포자의 단핵(monokaryon)과 이핵(dikaryon) 균사간의 차이를 보기 위한 기초실험이며, 또한 육종 계획에 대한 응용을 위한 실험이다.

• 한국동물위생학회지
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• 제37권4호
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• pp.247-252
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• 2014

The economic impact of swine mycoplasma infection is high. An accurate diagnosis is often difficult and time consuming. We report the development and validation of an effective multiplex polymerase chain reaction (PCR) assay that detects Mycoplasma (M.) hyopneumoniae and M. hyorhinis. The multi detection of M. hyopneumoniae and M. hyorhinis primer set were employed to detect mycoplasma species and typing of the species was performed on the basis of sequence analysis of the PCR product. The target nucleic acid fragments were specifically amplified by M. hyopneumoniae and M. hyorhinis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were used to evaluate the specificity of the multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. hyopneumoniae and M. hyorhinis.

• 한국응용곤충학회지
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• 제51권4호
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• pp.365-370
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• 2012

콩과(Fabaceae) 작물 해충인 팥나방(Matsumuraeses phaseoli)과 어리팥나방(M. falcana) (나비목: 잎말이나방과)은 형태적으로 매우 유사하여 종 구별이 힘든 것으로 알려져 있다. 본 연구에서는 PCR-SSP(PCR with Sequence Specific Primers) 방법으로 두 종을 빠르고 정확하게 구별할 수 있는 판별법을 찾고자 두 종의 미토콘드리아 시토크롬 옥시다제 I(mtCOI) DNA 부분영역(439 bp)의 염기서열을 해독하였다. 그리고 다른 나방 종의 mtCOI 염기서열과 함께 나열하여 비교한 후 팥나방과 어리팥나방에서 종 특이적으로 차이가 나는 단일 뉴클레오티드를 프라이머의 3' 말단으로 하는 염기서열 특이 프라이머 조합을 만들었다. PCR 산물들을 전기영동 한 결과, 어리팥나방은 245 bp, 팥나방은 409 bp와 245 bp의 특이적 밴드 패턴을 보여 두 종을 구별할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.490-495
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• 2007

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.