• The Plant Pathology Journal
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• 제21권3호
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• pp.229-236
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• 2005

Molecular profIles of PCR-RFLP and sequence analysis of internal transcribed spacer (ITS) regions of rDNA were compared between morphologically distinguishable species of Trichoderma isolated from substrates of oyster mushroom in Korea, T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum, T. virens, and two unidentified species, Trichoderma sp. 1 and 2. PCR­RFLP analysis divided the Trichoderma spp. into six RFLP groups, A, B, C, D, E, and F. The RFLP groups were generally agreed with described morphological species, except that the RFLP group A containing the two unidentified species. A neighbor-joining tree based on ITS sequences well supported RFLP groups observed by RFLP analysis of ITS regions of rDNA. Additionally, the two unidentified species, Trichoderma sp. 1 and 2, which could not be distinguished by PCR­RFLP analysis, were separated in sequence analysis of ITS regions of rDNA.

• 대한의생명과학회지
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• 제9권3호
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• pp.139-144
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• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• 대한수의학회지
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• 제43권1호
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• pp.67-75
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• 2003

A total of 260 dogs were randomly selected from two different treed kennels that brucellosis has occurred (group 1, 126 dogs), and random selected breed kennel (group 2, 134 dogs), and monitored for Brucella canis (B. canis) by 2-mercaptoethanol rapid slide agglutination test (2ME-RSAT) and bacterial culture method. For the differentiation, PCR-RFLP using omp-31, wbkA and per genes used for 52 of B canis strains (strain I) isolated in this study and 3 of B. canis strains (strain II) isolated in 1994 in Korea. 2ME-RSAT revealed that 63/126 dogs (50.0%) and 12/134 dogs (9.0%) were positive in group I and group II, respectively. Bacterial culture revealed that 47/126 dogs (37.3%) and 5/134 dogs (3.7%) were positive in group I and group II, respectively. As the results of PCR-RFLP, $\underline{omp}-31$ was amplified from all Brucella spp, except B. abortus. All B. canis isolates showed unique PCR-RFLP pattern following digestion with Bmel8I. However, all Brucella spp. showed the same PCR-RFLP pattern following digestion with SalI. PCR-RFLP analysis of wbkA revealed that all Brucella spp. showed the same pattern following digestion with HindIII. PCR-RFLP analysis of per revealed that B. abortus 544 and B. melitensis 63/9 showed the same pattern, but different from B. suis and B. canis following digestion with HindIII.

• 대한수의학회지
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• 제36권3호
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• pp.627-633
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• 1996

To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

• 대한의생명과학회지
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• 제5권2호
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• pp.209-212
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• 1999

라임병의 원인균인 Borrelia burgdorferi에 대하여 각 균종의 표준균주와 진드기에서 추출한 DNA를 template로 PCR을 실시한 후 그 증폭산물을 Alu I으로 처리한 restriction fragment length polymorphism (RFLP) 방법으로 각 균종의 연관성을 조사하고자 하였다. 표준균주로 RFLP를 실시한 결과 B. burgdorferi sensu stricto와 B. garinii의 RFLP 형태 (50 bp, 70 bp, 150 bp)가 유사하였으며 B. afzelii에서는 다른 RFLP 형태 (50 bp,110 bp,150 bp)를 관찰하였다. 그 중 B. afzelii KK-1과 B. garinii HPI은 새로운 RFLP형태를 보여 B. afzelii자 B. garinii는 각각 2 type의 subgroup으로 분류할 수 있었다. 진드기 DNA에서는B. afzelii를 포함한 각 균종에 대하여 모두 유사한 RFLP형태를 보였는데, 진드기 DNA에서 확인된 B. afzelii는 KK-1과 같은 군에 속하는 것으로 사료되었다.

• 생명과학회지
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• 제22권2호
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• pp.245-250
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• 2012

Vibrio속의 core 균주(Vibrio alginolyticus, Vibrio parahaemolyticus)를 포함하여 총 6 종의 Vibrio 균주(V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus)와 Grimontia (Vibrio) hollisae의 16S rDNA를 PCR 증폭하여 Alu I, Cfo I, Dde I, Hae III, Msp I, Rsa I의 6 종의 제한효소를 처리 후 RFLP 분석을 수행하였다. 2 종의 core 균주와 V. proteolyticus는 4 종의 제한효소(Cfo I, Dde I, Msp I, Rsa I)에서 동일한 제한효소 패턴을 나타내었다. 제한효소의 패턴의 조합에 의해 6 종의 Vibrio 종은 6 개의 RFLP type으로 구분되었다. 특히 Alu I의 경우, 실험된 6 종의 Vibrio속에 대하여 각기 다른 6 개의 종 특이적 RFLP type을 나타내었다. 제한효소 패턴에 근거하여 작성한 덴드로그램에서 Vibrio core group 균주인 V. alginolyticus 와 V. parahaemolyticus는 90% 이상의 매우 높은 유사도를 나타내었다. 반면 Grimontia hollisae는 실험된 모든 제한효소 패턴에서 Vibrio속 세균과는 분명히 구분되는 RFLP type을 나타내었다. 따라서 PCR-RFLP는 제한효소를 적절히 선택한다면 Vibrio 속 세균의 신속한 구분에 여전히 유용하다.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• 식물분류학회지
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• 제41권2호
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• pp.119-129
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• 2011

한국산 초롱꽃과 8속 25분류군과 군외군 2분류군 등 총 27분류군에 대한 계통유연관계를 알아보기 위하여 PCR-RFLP와 ITS 지역의 염기서열 분석을 실시하였다. 엽록체 DNA의 11개 지역을 이용한 PCR-RFLP 분석에서는 증폭된 DNA 각각에 대하여 15가지 제한효소를 처리한 결과 총 244개의 제한효소 절편을 얻었으며, 그 중 59개는 분류군간 차이가 있어 24%의 다형화를 보였다. ITS 지역의 길이는 588-707 bp이었으며, 염기변이는 0-39.36%로 나타났다. 계통 분석 결과, PCR-RFLP와 ITS 지역의 염기서열을 이용한 계통수 모두에서 초롱꽃과는 단계통을 형성하였다. 도라지속과 더덕속은 독립적인 분계조로 유집되었고, 홍노도라지속과 영아자속은 잔대속-금강초롱꽃속을 위한 자매군을 형성하였다. 초롱꽃속도 단계통으로 나타났고, 애기도라 지속은 ITS분석에서는 가장 기부에 분계조를 형성하였지만 PCR-RFLP결과에서는 초롱꽃속과 더덕속-도라지속분계조 사이에 위치하여 차이를 보였다. 금강초롱꽃속은 잔대속과 함께 유집되었으며, 잔대속은 병계원으로 분계조를 형성하여 형태형질에 의한 속내 분류체계와 일치하지 않았다. 본 연구에서 다룬 2가지 자료에 기초한 한국산 초롱꽃과의 계통분석 결과, 속 수준에서는 대부분 일치하는 경향을 보였지만 애기도라지속과 금강초롱꽃속의 계통학적 위치와 잔대속의 속내 분류계급의 유집에서는 차이를 보였다.

• Fisheries and Aquatic Sciences
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• 제7권3호
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• pp.130-135
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• 2004

The marine dinoflagellate genus Alexandrium, which produces PSP toxins, has a global distribution. As human-assisted dispersal of the species has been suggested, it is important to develop molecular tools to trace the dispersal pathway. To screen population-specific DNA sequences that differentiate Korean and Japanese A. catenella, we targeted the area downstream of the chloroplast psbA gene using PCR with population-specific DNA primers followed by RFLP (restriction fragment length polymorphism) analysis and sequencing. The RFLP patterns of the PCR products divided Korean and Japanese A. catenella regional isolates into three types: Korean, Japanese, and type CMC3, isolated from Korea. We sequenced the PCR products, but found no similar gene in a homology search. The molecular phylogeny inferred from the sequences separated the Korean and Japanese A. catenella strains, as did the RFLP patterns. However, the Japanese isolates included two slightly different sequences (types J and K), while the Korean sequence was the same as the Japanese K type. In addition, a unique sequence was found in the Korean strains CMC2 and CMC3. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers designed from the type J sequence yielded PCR products for Japanese strains only, showing that the unknown gene can be used for a population analysis of Korean and Japanese A. catenella.

• Clinical and Experimental Pediatrics
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• 제48권10호
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• pp.1068-1075
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• 2005

목 적 : 성장호르몬 분비를 촉진하는 ghrelin는 여러 질병에서의 역할은 아직까지 잘 알려져 있지 않았다. 그러나 최근에 ghrelin 유전자 변이가 비만과 당뇨병과 관련이 있는 것으로 알려졌다. 현재까지 보고된 ghrelin 유전자 이상으로는 SNP인 Arg51Gln, Leu72Met, G274A가 있으며 이중 가장 흔한 이상인 Leu72Met 변이이다. 일반적으로 ghrelin 유전자의 Leu72Met와 같은 diallelic 다형성을 스크리닝하는데 SSCP, RFLP, sequencing 등이 사용되어 왔으나 향후 비만 환자들에서 가장 효과적이고 정확하게, 손쉽게 ghrelin 유전자의 Leu72Met 다형성 분석을 검출할 수 있는 스크리닝 방법을 찾아 임상적 적용에 이용하고자 하였다. 방 법 : 비만소아에서 ghrelin 유전자의 Leu72Met 다형성 분석을 PCR-RFLP, PCR-SSCP와 ARMS 분석 방법을 이용하여 비교분석하고 이들의 검사방법의 장단점을 알아보았다. 결 과 : PCR-RFLP, PCR-SSCP와 ARMS 분석 모두에서 allele 특이산물의 밴드가 뚜렷이 구별할 수 있었으며 상기 방법에 의한 결과는 모두에서 일치하였다. PCR-SSCP 방법은 점돌연변이의 존재 여부를 많은 시료를 대상으로 쉽게 분자학적 스크리닝할 수 있는 장점이 있으나 조작이 간단하지 않고 비용부담이 많다. BsrI 제한효소를 이용한 PCR-RFLP법은 비교적 용이하고 간단하여 널리 쓰이는 방법이지만 충분한 고농도의 DNA가 필요하며 전기영동 결과의 올바른 해석이 쉽지 않았다. 이에 비하여 ARMS 분석법은 위의 방법들보다는 간편하여 검사의 소요시간도 짧으며, 검출률이 높고 결과 해석이 매우 쉬웠다. 결 론 : 따라서 본 실험에서 시행한 ghrelin의 Leu72Met 유전자의 다형성을 결정하는 방법 중에는 ARMS 분석법이 정확하고 검사 분석법 및 결과 해석이 매우 쉽고 검사의 소요시간도 짧아 대량 검체에 적용에 매우 효과적으로 사료된다.