• 식물병연구
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• 제18권2호
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• pp.73-79
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• 2012

Phytophthora katsurae는 밤나무 잉크병의 원인이 되는 병원성 균류이다. P. katsurae를 검출하기 위하여 2개의 duplex PCR primer set을 제작하였다(SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R). SOPC 1F/1R과 SOPC 1-1F/1-1R primer pair는 sequence characteristic amplification regions(SCAR)를 이용하여 제작하였고, KatI 3F/5R primer pair는 internal transcribed spacer(ITS) 부위로부터 제작하였다. 추출한 genomic DNA를 1 mg/ml에서 1 ng/ml까지 10배씩 순차적으로 희석하여 균사량에 따른 Duplex PCR의 민감도를 확인한 결과, 2개의 primer set은 각각 1 ${\mu}g/ml$와 100 ng/ml까지 검출이 가능하였다. 유주포자를 $1{\times}10^6$부터 $1{\times}10^2$ cells/ml까지 10배씩 순차적으로 희석하고 genomic DNA를 추출하여 P. katsurae의 유주포자량에 의한 검출 한계를 확인한 결과, 2개의 primer set 모두 $1{\times}10^5$ cells/ml까지 검출할 수 있었다. 각각의 primer set은 PCR 검출을 실시하였을 때 P. katsurae 균주에서만 PCR 산물이 증폭된 반면에, Phytophthora 16종에서는 P. katsurae에 특이적인 PCR 증폭산물이 생성되지 않았다. 따라서, 2개의 primer set을 이용한 Duplex PCR은 P. katsurae의 신속하고 효과적인 검출에 유용하게 활용될 수 있을 것이다.

• 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.

• 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.

• Mycobiology
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• 제41권4호
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• pp.252-255
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• 2013

Fungal pathogens have caused severe damage to the commercial production of Pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. Four strains of pathogenic fungi, including Trichoderma koningiopsis DC3, Phomopsis sp. MP4, Mucor circinelloides MP5, and Cladosporium bruhnei MP6, were isolated from the bottle culture of diseased P. eryngii. A species-specific primer set was designed for each fungus from the ITS1-5.8S rDNA-ITS2 sequences. PCR using the ITS primer set yielded a unique DNA band for each fungus without any cross-reaction, proving the validity of our method in detection of mushroom fungal pathogens.

• 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.

• Mycobiology
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• 제29권1호
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• pp.7-10
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• 2001

Based on the rDNA ITS sequences data, specific primer set for PCR detection of wood-decaying fungus Phellinus linteus was designed. The length of PCR products using designed primer set(SHF and SHR) was about 540 bp. Among 11 species, 17 isolates of Phellinus spp. including Phellinus linteus, P. pomaceus, P. spiculosus, P. baumi, P. pini, P. igniarius, P. gilvus, P. biscuspidatus, P. weirii, P. johnsonianus, P. robutus, and P. igniarius, seven isolates of Phellinus linteus showed about 540 bp-sized single band. This molecular technique could offer a useful tool for detecting and identifying Phellinus linteus.

• The Plant Pathology Journal
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• 제24권1호
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• pp.17-23
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• 2008

A total of 33 isolates of Colletotrichum species obtained from pepper, apple, and strawberry in 2001 and 2002 were identified based on mycological characteristics, responses to fungicides carbendazim and the mixture of carbendazim and diethofencarb, and nucleotide sequence analysis of internal transcribed spacer (ITS) regionMost of the Colletotrichum isolates from pepper could be identified as C. acutatum. The pepper isolates produced grey white mycelia that gradually changed to dark gray. The conidia were variable in size, and almost cylindrical in shape with at least one rounded end. They could grow on PDA amended with carbendazim or with the mixture of carbendazim and diethofencarb at $10{\mu}g/ml$, to which the isolates from apple and strawberry were very sensitive. A part of the ITS regions from the Colletotrichum isolates was amplified with the specific primers designed for C. acutatum (Ca1-1) or C. gloeosporioides (Cg1-3). A primer pair of Ca1-1 and a universal primer (ITS4) amplified a 496-bp DNA fragment from all of the pepper isolates examined and one apple isolate. Taken together, it is conclusive that the Colletotrichum isolates causing the typical lesion of anthracnose on pepper fruits are C. acutatum.

• Food Science and Biotechnology
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• 제17권2호
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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.

• 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.

• Mycobiology
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• 제33권2호
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• pp.104-108
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• 2005

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.