• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.91-98
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• 1996

벼도열병균 14개 균주와 벼 이외 화본과 식물 도열병균 12개 균주를 대상으로 rDNA의 ITS II 부위를 증폭하여 그들의 핵산 구조 차이를 분석함으로 도열병균 균주간 분류를 시도하였다. 5.8S rDNA의 3`-말단 부위와 28S rDNA의 5`-말단 부위의 sequence 중 5`-CCCGGGAATTCGCATCGATCGATCGAATGAAGA-ACGCAGC-3`와 5`-CCCGGGATCCTCCGCTTATT-GATATGC-3`를 이용하여 PCR 증폭을 하였을 때 벼도열병균 14개 균주는 동일한 길이의 단일 밴드를 형성하였으며 벼 이외 화본과 식물 도열병균에서는 레드톱 식물로부터 분리한 도열병균만이 나머지 균주보다 38bp가 더 큰 길이를 가진 밴드를 형성하였다. PCR로 증폭된 DNA를 HaeIII와 MspI 제한효소로 절단하였을 때 벼도열병균 레이스간에는 제한효소 절단에 의한 전기영동 밴드 형태 차이를 관찰할 수 없었으나, 벼 이외 화본과 식물 도열병균 12개 균주는 3군으로 구분할 수 있었다. 벼도열병균 90=054와 강아지풀에서 분리한 도열병균 G90-5, 기장에서 분리한 G88-4, 바랭이에서 분리한 G88-5 그리고 레드톱에서 분리한 RT 균주의 ITS II 부위의 DNA 염기서열 비교 분석에 의하면 G88-4와는 다른 HaeIII와 MspI 제한효소 위치를 가지고 있었기에 제한효소 절단에 의한 전기영동 형태가 상이하였다. 또한 RT균주는 HaeIII와 MspI위치가 존재하지 않았다.

• The Korean Journal of Mycology
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• v.45 no.4
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• pp.386-393
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• 2017

Onion downy mildew, caused by Peronospora destructor, is a major disease in onion cultivation areas in Korea. The causal fungi were collected and analyzed based on sequence similarity and molecular phylogenetic relationships of multi-gene sequences, including the internal transcribed spacer (ITS) region. All isolates from Changnyeong-gun, Hamyang-gun, and Hapcheon-gun in Gyeongnam province, and Muan-gun, Haenam-gun, and Sinan-gun in Jeonnam province were identical in the four types of gene sequences, indicating they were genetically the same strains. In this study, a PCR method was developed based on the ITS gene sequences to amplify the specific DNA fragment for P. destructor only. The detection limit of was total genomic DNA of the P. destructor and the plant $0.7ng/{\mu}L$. Therefore, the developed PCR method could be used to detect P. destructor effectively from symptomless onion leaves.

• Parasites, Hosts and Diseases
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• v.45 no.1 s.141
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• pp.69-74
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• 2007

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction(PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each(520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

• The Korean Journal of Mycology
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• v.49 no.4
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• pp.455-467
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• 2021

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH₄)₂SO₄ after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

• Research in Plant Disease
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• v.17 no.2
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• pp.169-176
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• 2011

Phytophthora katsurae is the fungus responsible for chestnut ink disease. The objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis of rDNA-ITS region, elongation factor 1 alpha gene and ${\beta}$-tubulin gene could be used for rapid identification and genetic diversity of P. katsurae, and to assess the potential use of the SSCP technique as a diagnostic tool for P. katsurae. Each regions amplified by PCR using primers designed to overlap the genus Phytophthora were characterized for the Phytophthora species. PCR products were denatured and electrophoresed for SSCP analysis. P. katsurae isolates showed an unique pattern in SSCP analysis and were easily distinguished from other Phytophthora species used as the control. This indicates that SSCP analysis is an useful technique for distinguishing Phytophthora species from genetically close relatives, and show that the SSCP analysis of each region is an efficient detection tool for P. katsurae. But PCR-SSCP analysis of single-gene may have difficulty in distinguishing P. katsurae from other Phytophthora species. Therefore, PCR-SSCP analysis of multi-genes can be useful for rapid and effective identification of P. katsurae.

• Research in Plant Disease
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• v.27 no.3
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• pp.120-127
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• 2021

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

• Fisheries and Aquatic Sciences
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• v.11 no.1
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• pp.32-35
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• 2008

The Suminoe oyster, Crassostrea ariakensis, occurs in estuaries where rivers meet seawater. In Korea, it is one of the most popular fisheries resources in the Nam River and Sumjin River. However, the genetic identification of this species has been questioned, because specimens are often mis-identified as other species. To identify the species, we conducted polymerase chain reaction (PCR) amplification of the internal transcribed spacer-1 (ITS-1) region, followed by digestion with the restriction enzyme HaeIII. Restriction profiles for oysters collected from Korea, Japan, and China (north and south) were determined by comparing the PCR-restriction fragment length polymorphism (RFLP) patterns of the ITS-1 regions. Our study verified that the oysters collected from Korea were C. ariakensis based on the PCR-RFLP patterns. These results emphasize the value of molecular markers for identifying morphologically uncertain species.

• Journal of Environmental Science International
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• v.18 no.12
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• pp.1331-1338
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• 2009

Marine dinoflagellate Alexandrium minutum producing paralytic shellfish toxins is responsible for paralytic shellfish poisoning (PSP). To investigate its temporal distributions in Chinhae Bay where PSP occurs annually, SYBR Green I based A. minutum-specific real-time PCR probe was developed on the LSU rDNA region. Assay specificity and sensitivity were tested against related species, and its specificity was further confirmed by sequencing of field-derived samples. Ten months field survey in 2008 (a total 100 surface water samples) by using the real-time PCR probe showed that A. minutum was detected at very low densities of 1-4 cells $L^{-1}$ in May and June being spring in Chinhae Bay, Korea.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.589-593
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• 1998

DNA amplification by polymerase chain reaction (PCR) was used to specifically detect Plasmodiophora brassicae, causing clubroot of crucifers. On the basis of DNA sequence informations, an oligonucleotide primer set specific for the pathogen was designed form small subunit gene (18S-like) and internal transcribed spacer (ITS) region of ribosomal DNA. Primer ITS 5/PB-C produced an amplification product of approximately 520 bp in length with DNA from P. brassicae. However, no amplification product was produced with DNAs from several soil-borne fungi, Didymella bryoniae and Rhizopus stolonifer. Using these primers, the clubroot pathogen was readily detected from infected roots of crucifers, but not from healthy roots. Southern hybridization analysis further confirmed that the amplification product was originated from P. brassicae.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.357-361
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• 2008

One of the major allergenic proteins in common buckwheat (Fagopyrum elculentum) was found to be a BW10KD. In this work, allergenic BW10KD genomic DNAs from the native common buckwheat 'Pyeongchang' and Tartarian buckwheat 'Clfa47' were cloned by polymerase chain reaction (PCR), and their nucleotide sequences were determined. In addition, a novel PCR assay targeting the allergenic BW10KD gene was developed to detect and differentiate both buckwheat species in food. The nucleotide sequences of the BW10KD genomic DNA from 'Pyeongchang' and 'Clfa47' were 94% identical. Base differences in the nucleotide sequences of the BW10KD genes are probably useful as a molecular marker for species-specific identification. The 'Pyeongchang'-specific primer set 154PF/400PR and the 'Clfa47'-specific primer set 154DF/253DR generated 247 and 100 bp fragments in singleplex PCR, respectively. A duplex PCR assay with 2 species-specific primer sets simultaneously differentiated the 'Pyeongchang' and 'Clfa47' in a single reaction. The PCR assay also successfully allowed for the rapid detection of buckwheat ingredients in foods.