• Journal of Microbiology
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• 제45권4호
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• pp.297-304
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• 2007

The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1170-1177
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• 2005

Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor inoculated oil-degrading microorganisms during bioremedial treatability tests. A pair of universal primers, fluorescently labeled 521F and 1392R, was employed to amplify small subunit rDNA in order to simultaneously detect two bacterial strains, Corynebacterium sp. IC10 and Sphingomonas sp. KH3-2, and a yeast strain, Yarrowia lipolytica 180. Digestion of the 5'-end fluorescence/labeled PCR products with HhaI produced specific terminal-restriction fragments (T-RFs) of 185 and 442 bases, corresponding to Corynebacterium sp. IC10 and Y. lipolytica 180, respectively. The enzyme NruI produced a specific T-RF of 338 bases for Sphingomonas sp. KH3-2. The detection limit for oildegrading microorganisms that were inoculated into natural environments was determined to be $0.01\%$ of the total microbial count, regardless of the background environment. When three oil-degrading microorganisms were released into oil-contaminated sand microenvironments, strains IC10 and 180 survived for 35 days after inoculation, whereas strain KH3-2 was detected at 8 days, but not at 35 days. This result implies that T-RFLP could be a useful tool for monitoring the survival and relative abundance of specific microbial strains inoculated into contaminated environments.

• 대한미생물학회지
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• 제35권1호
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• pp.49-60
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• 2000

ITSI-5.8S-ITSII rDNA region was amplified from the reference strains and clinical isolates with ITS1 and ITS4 primers. These primers amplified DNA fragments of 550 bp in Microsporum audouinii and Trichophyton violaceum, 700 bp in Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton tonsurans, and 750 bp in Microsporum ferreugineum and Microsporum canis. The restriction enzyme patterns of PCR products digested with 13 restriction enzyme including PstI were distint among the genera, whereas identical in the same species. Examination of the ITS (Internal Transcribed Spacers)1 nucleotide sequence revealed that there was the genetic difference in each genera and species. Phylogenetic relationship among each species showed that the Trichophyton mentagrophytes was more closely related Trichophyton tonsurans than Trichophyton rubrum, and Microsporum gypseum was less related than Microsporum spp..

• 생명과학회지
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• 제15권6호
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• pp.968-971
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• 2005

본 연구는 Duroc, Landrace, Berkshire, Yorkshire를 기초 축으로 이용한 1003두에 대해 MC4R유전자의 PCR-RFLP를 이용하여 그 다형성을 조사하고 돼지의 일당증체량, 등지방 두께, 사료 요구율, 정육율과 그 유전자형 간의 연관성을 규명하고자 실시하였다. MC4R유전자에 대해 PCR-RFLP를 이용하여 226bp산물을 증폭한후 Taq I 체한효소로 사용하였다. 얻어진 MC4R gene의 유전자 빈도는 품종별로 다르게 나타났다. 통계적 분석을 통하여 각 유전자형에 대한 경제형질과 관련성을 분석한 결과 일당 증체량과 사료요구량은 NN 유전자형을 가진 개체들이 DN이나 DD유전자형을 가진 개체들에 비해 유의적으로 우수한 능력을 보였다(P < 0.05). 하지만 D 대립유전자는 높은 정육율과 낮은 등지방두께에 연관성이 있음을 관찰하였다. 따라서 돼지의 성장과 정육율과 관련된 선발력을 높이기 위해서 MC4R유전자의 다형성분석에서 검증된 PCR marker를 우량돼지육종 계획에 있어 분자생물학적 선발 marker로 사용할 수 있을 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제35권4호
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• pp.277-282
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• 1997

만손열두조충과 북미열두조충 (Spirometra mansonoides)의 형태 차이점은 성숙편절의 자궁의 형대 가 전자근 차곡차곡 쌓인 꼴이고 후자는 알과벳 씨자 (C)형태이라는 점이다 이 비슷한 명태의 두 종 의 조충이 유전학적으로는 얼마나 차이가 있는지를 알기 위하여 중합효소연쇄반응-마디길이여러꼴 분석법(polymerase chain reaction-restriction fragment length polymorphism analysis)을 이용하여 유전 형질을 비교하였다. 충체로부터 285리넓솜 리보핵산 (285 rDNA), 사립체 cytochlmsc 산화 효소 아단위 I (mitochondrial cytochrome c oxidase subunit 1, mCOI) 및 리보솜 내부 전사된 영역 1 (ribosomal internal transcribed spacer 1, ITSI)에 대한 중합효소반응 산물을 구하였다. 이 산를은 Msp I. Hue III, Alu I, Cfo I, Rsc I의 4 염기 제한 효소로 잘라서, 전기영동하여 길과를 PAUP 3.1.1을 이용하여 분석 하였다. 285 리보솜 리보핵산과 리보솜 내부 전사된 영역 1 유진자에서는 두 충체 가 동일 한 분류가지에 묶였고 홀로서기수 (bootstrap number)는 94, 100이었다. 사립체 cytochrome c 산 화효소 아단위 1에서는 다른 분류가지에 묶였고 홀로서기수가 74이었다. 위 결과로 투 조충이 같은 조상에서 유래함과 진화 단계에서 매우 가까운 위치에 있음을 알 수 있었다.

• Parasites, Hosts and Diseases
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• 제37권2호
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• pp.121-125
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• 1999

Inoculation of human fecal cysts to suckling Mongolian gerbils, two Giardia lamblia isolates, Kl and K2, were established as axenic cultures. Using this in vitro culture, both two Giardia isolates were grouped by using two genetic analysis. With genetic analysis of SSS-rDNA sequences, both K1 and K2 were found as members of hopkins'group 1, despite some nucleotide differences noticed in K1 (5 differences/292 bases.). The other genetic study used PCI-RFLP of the tim (triose phosphate isomerase) Nash's group 2 can bot be a separate group, but a part of Hopkins' group 1.

• BMB Reports
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• 제34권2호
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• pp.114-117
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• 2001

Cervi Parvum Cornu is widely used as a hemopoietic, tonifying, growth-promoting, cardiotonic, and immuno-modulating agent in Korea. In order to develop the quality control method of Cervi Parvum Cornu by the identification of the biological source or origin, the molecular approach was applied using PCR (polymerase chain reaction) and PCR-RFLF (PCR-restriction fragment length polymorphism) analysis. In the PCR analysis of the mitochondrial 12S rRNA gene and cytochrome b gene regions, no distinctive DNA bands from Cervidae (deer) antlers and Rangifer (reindeer) antlers were observed. However, when the amplified products in the mitochondrial cytochrome b gene region were subjected to restriction digestion with TaqI, Cervidae antlers showed an undigested state of 380 by band, differently from two bands of 230 by and 1S0 by from Rangifer antlers. Based on this finding, the base sequences of amplified PCR products in the range of mitochondria) cytochrome b gene from Cervidae antlers and Rangifer antlers were determined and subjected to restriction analysis by various endonucleases. The results showed that antlers from Rangifer species could be simply discriminated with other antlers from 8 Cervidae species (Chinese deer, Russian deer, Hong Kong deer, New Zealand deer, Kazakhstan deer, elk, red deer and Sika deer) by PCR-RFLP analysis using AtuI, HaeIII, HpaII or Sau3AI(MboI) as well as TaqI in the range of the mitochondrial cytochrome b gene.

• 한국해양학회지:바다
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• 제15권1호
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• pp.25-35
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• 2010

플랑크톤의 종다양성은 특정 지역의 수계환경 변화 모니터링에 있어 중요 생태지표로써, 환경 평가에 유용한 정보로 사용되고 있다. 기존의 종다양성 평가는 주로 형태학적 형질에 근거한 종동정을 통해 이루어졌으나, 많은 시간과 전문성을 필요로 하고 연구자의 주관적 판단에 의존하는 단점이 있다. 따라서, 본 연구에서는 채수된 환경시료에 대해 보다 빠르고 정확한 플랑크톤 종다양성을 파악하기 위하여 분자마커를 활용한 분자모니터링 기법을 도입하였다. 서낙동강(김해교)과 남해 연얀(남해도) 정점에서 각각 채수된 환경시료에서 DNA를 추출한 후 18S nuclear ribosomal RNA 유전자를 대상으로 중합효소연쇄반응을 수행하였다. 클로닝 과정을 통해 만들어진 각각의 클론 라이브러리에서 클론을 무작위로 선택하여 제한효소절편다형성 패턴분석을 한 후 특이성을 가지는 클론을 선별하였다. 김해교에서는 60개 블론을 대상으로 44개의 특이적 클론을 선별하였고 남해에서는 150개 클론을 대상으토 27개의 클론을 선별하였다. 이틀 클론틀에 대한 염기서열 분석결과 다양한 계통분류군에 속승하는 플랑크톤의 종조성 결과를 보여주었다(김해교: Heterokontophyta(7), Ciliophora(23), Dinophyta(l), Chytridiomycota(l), Rotifera(I), Arthropoda (11), 남해: Ciliophora( 4), Dinophyta(3), Crγptophyta(l)，Arthropoda(19)). 본 연구를 통하여 분자마커를 활용한 분자모니터링 기법이 기존 형태학적 형질에 근거한 분석이 가지는 한계를 보완하여 채수된 환경시료의 종조성 분석에 효율적으로 사용될 수 있다고 판단된다.

• Toxicological Research
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• 제32권1호
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• 한국식물병리학회지
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• 제14권5호
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• pp.452-457
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• 1998

Thirty-eight isolates of Phytophthora sp. caused rots on roots and basal stems were collected from five flowering plants from 1992 to 1997 at eight cultivation areas in Korea. All the isolates were identified as P. nicotianae based on following characteristics. The fungus produced markedly papillate, not caducous and ovoid to spherical sporangia, abundant chlamydospores, and small oospores with amphigynous antheridia only when paired with either A1 or A2 mating type. All isolates grew well at 35$^{\circ}C$ and showed distinct arachnoid colony patterns on CMA and PDA. Sizes of sporangia and chlamydospores of five representative isolates from each plant averaged 43-52$\times$30-38 ${\mu}{\textrm}{m}$ and 28 ~34 ${\mu}{\textrm}{m}$. Mating type of the isolates was either A1 or A2, and oogonia and oospores were measured as 28~31 ${\mu}{\textrm}{m}$ and 21~25 ${\mu}{\textrm}{m}$. PCR-RFLP analysis of rDNA of the five isolates resulted that restriction band patterns of the small subunit and ITS regions were identical to a perilla isolate of P. nicotianae, but distinct from P. cactorum and P. capsici. Cross inoculation tests showed that the five isolates had pathogenicity to lily, christmas cactus, anthurium, baby's breath and carnation with different degrees. However, each isolate showed stronger pathogenicity to its corresponding original host than others. Among five lily cultivars Georgia and Quririna were more susceptible than Napoli and others. This is first report of Phytophthora root and stem rot of lily, Christmas cactus, anthurium, baby's breath and monochoria in Korea.