• The Plant Pathology Journal
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• v.21 no.3
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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.

• Biomedical Science Letters
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• v.9 no.3
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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.

• Korean Journal of Veterinary Research
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• v.43 no.1
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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.

• Korean Journal of Veterinary Research
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• v.36 no.3
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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.

• Biomedical Science Letters
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• v.5 no.2
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• pp.209-212
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• 1999

For the classification of B. burgdorferi sensu lato strains, PCR-restriction fragment length polymorphism (RFLP) analysis was performed. PCR was carried out with B. burgdorferi sensu lato specific primer set (BB uni set), and amplicons of 470-bp DNA were digested with Alu 1. The Alu I restriction polymorphism of the amplicons provided a useful tool for identifying B. burgdorferi sensu late strains. Both amplicons from B. burgdorferi sensu stricto and B. garinii except HPI strain showed identical RFLP pattern (50 bp, 70 bp, and 150 bp), but amplicons from B. afzelii and B. garinii showed two types of subgroups, respectively. The result of PCR-RFLP using extracted DNAs from ticks was similar to those patterns of B. burgdorferi species including B. afzelii.

• Journal of Life Science
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• v.22 no.2
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• pp.245-250
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• 2012

The 16S rDNA - RFLP types for six Vibrio species (V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus) including two core group members, V. alginolyticus and V. parahaemolyticu s, and Grimontia (Vibrio) hollisae were determined using PCR-RFLP analysis. Six tetrameric restriction enzymes (Alu I, Cfo I, Dde I, Hae III, Msp I, and Rsa I) were selected for RFLP analysis. V. alginolyticus, V. parahaemolyticus, and V. proteolyticus showed the same RFLP pattern following digestion with four of the six used restriction enzymes: CfoI, DdeI, MspI, and RsaI. Various restriction enzyme combinations generated digests recognizable as distinct RFLP types for each of the assayed Vibrio species. In particular, AluI single digestion produced species specific band patterns that enabled the differentiation between these Vibrio species. Dendrogram based on restriction patterns showed that two Vibrio core group members, V. alginolyticus and V. parahaemolyticus were closely related having a similarity over 90%. Although the observed RFLP pattern for Grimontia hollisae shared several common bands with other Vibrio spp., G. hollisae results were still clearly distinct from Vibrio spp. RFLP types for all restriction enzymes tested. If restriction enzymes are aptly selected, PCR-RFLP analysis is still a rapid and effective tool for differentiating Vibrio species.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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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.

• Korean Journal of Plant Taxonomy
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• v.41 no.2
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• pp.119-129
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• 2011

Phylogenetic studies were conducted to evaluate the taxonomic relationships among 27 taxa, including 2 outgroups of Korean Campanulaceae, using PCR-RFLP analysis and ITS sequences. In the PCR-RFLP analysis, 15 restriction endonucleases produced 244 restriction sites and size variations from the chloroplast DNA, and 59 restriction sites (24%) showed polymorphism. The length of the ITS regions ranged from 588 bp to 797 bp. The sequence divergence including the outgroups is 0-39.36%. Phylogenetic analyses based on PCR-RFLP and ITS data suggest that Campanulaceae is monophyletic; Codonopsis and Platycodon forms an independent clade; the Peracarpa and Asyneuma clade is a sister to the Adenophora-Hanabusaya clade; Campanula is monophyletic; and Wahlenbergia basally branches within the ITS tree, whereas they are placed between Campanula and the Codonopsis-Platycodon clade in the PCR-RFLP tree; Hanabusaya is placed within the Adenophora clade; and Adenophora is paraphyletic and shows discordance to the infrageneric classifications based on morphological data. The present results show two data sets, largely congruent at the generic level, but their phylogenetic positions, in particular the Wahlenbergia and Hanabusaya and the infrageneric classifications in Adenophora, show some incongruence.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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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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• v.48 no.10
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• pp.1068-1075
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• 2005

Purpose : The role of ghrelin, which promotes the secretion of growth hormone, was not well known until now. Recently it was found that the mutation of ghrelin gene is related to obesity and diabetes. This study is to find the screening method that can easily and effectively detect the polymorphism of Leu72Met in ghrelin gene of obesity patients and apply it to clinical usage. Methods : We compared PCR-RFLP, PCR-SSCP and ARMS methodologies for analyzing of the polymorphism of Leu72Met in ghrelin gene of obesity children, and also studied the merits and demerits of these methodologies. Results : In this study, we were able to find out the band of peculiar allele of Leu72Met in ghrelin gene using PCR-RFLP, PCR-SSCP and ARMS analyses. The polymorphism of Leu72Met in ghrelin gene determined by all above methodologies was in complete agreement. Compared to the PCR-RFLP and PCR-SSCP, ARMS analysis is simple, inexpensive and also consume less time. It is very sensitive to analyze the polymorphism and easy to understand the results of test. Conclusion : Though PCR-RFLP, PCR-SSCP and ARMS analyses were sensitive to analyze the polymorphism of Leu72Met in ghrelin gene, ARMS analysis appears to be more efficient than PCR-RFLP and PCR-SSCP. Therefore, we conclude that ARMS analysis is suitable to analyze the polymorphism of Leu72Met in ghrelin gene for large quantity of specimens.