• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1368-1374
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• 2005

To investigate the genetic relationships among various cattle breeds, bovine mtDNA D-loop region was used in 411 animals of 18 cattle breeds, including 8 Asian Bos taurus, 7 European Bos taurus, 1 Asian Bos indicus, and 2 African Bos indicus. The size of amplified PCR products from mtDNA D-loop region was 964 bp and the products were digested by 15 different restriction enzymes. Two different band patterns were identified in eight restriction enzymes (BstXI, Hae III, Msp I, Apa I, Taq I, Alu I, BamH I, EcoN I) and the rest of restriction enzymes showed more than 3 different band patterns among which Apo I and MspR9 resulted in 7 different restriction patterns. The genotypes, number of haplotype, effective number of haplotype, and degree of heterozygosity were analyzed. Based on all the PCR-RFLP data, different haplotypes were constructed and analyzed for calculating genetic distances between these breeds using Nei's unbiased method and constructing a phylogenetic tree.

• Research in Plant Disease
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• v.23 no.3
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• pp.241-248
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• 2017

The purpose of this study was to develop rapid methods for distinguishing between Heterodera schachtii and H. glycines detected from chinese cabbage fields of highland in Gangwon, Korea. To do this, we performed PCR-RFLP and PCR with the primers set developed in this study for GC147, GC408 and PM001 population, H. schachtii, and YS224, DA142 and BC115 population, H. glycines. Eight restriction enzymes generated RFLP profiles of mtDNA COI region for populations of H. schachtii and H. glycines, repectively. As a result, treatment of two restriction enzymes, RsaI and HinfI, were allowed to distinguish H. schachtii from H. glycines based on the differences of DNA band patterns. The primer set, #JBS1, #JBG1 and #JB3R, amplified specific fragments with 277 and 339 bp of H. schachtii, 339 bp of H. glycines, respectively, while it did not amplify fragments from three root-knot nematodes and two root-lesion nematodes. Thus, the primer set developed in this study could be a good method, which is used to distinguish between H. schachtii and H. glycines.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.127-134
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• 1997

Transmission electron microscopy of an ArGnthnmoebo isolate (KA/LS) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acnnthamoebn isolate belonged to the morphological group ll. Based on the polymerase chain reaction (PCR) - restriction fragment leilgth polymorphism (RFLP) of 185 ribosomal RNA coding DNA (rDNA) , the isolate was identified as A. Iwnunensis. Strain typing by isoenzyme analysis using isorlectric focusing (IEF) and mitochondrial (Ent) DNA RFLP revealed that the isolate was closely related with KA/Ll , the most predominant type of isolates from contact lens storage casas, KA/E2, a clinical isolate, KA/W4, previou:fly reported to host endosymbionts. and L3a strains of A. Iwnunensis. The endosymbionts were similar to those of KA/W4 in a.jpects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteri3 measuring approximately 1.38 x 0.50 ㎛. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunae- like stnlcture.

• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.181-188
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• 1999

We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships. Twenty-five isolates representing 20 species were included in the analysis. As in nuclear 18s rDNA analysis, two type strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged earliest from the other strains, but the divergence between them was less than in 18s riboprinting. Acanthamoeba griffini of morhological group 2 branched between pathogenic (A. culbertsoni A-1 and A. healyi OC-3A) and nonpathogenic (A.palestinensis Reich, A. pustulosa GE-3a, A. royreba Oak Ridge, and A lenticulata PD2S) strains of morphological group 3. Among the remaining isolates of morphological group 2, the Chang strain had the identical mitochondrial riboprints as the type strain of A. hatchetti. AA2 and AA1, the type strains of A. divionensis and A. paradivionensis, respectively, had the identical riboprints as A. quina Vil3 and A. castellanii Ma. Although the branching orders of A. castellanii Neff, A. polyphaga P23, A. triangularis SH621, and A. lugdunensis L3a were different from those in 18S riboprinting analysis, the results obtained from this study generally coincided well with those from 18S riboprinting. Mitochondrial riboprinting may have an advantage over nuclear 18S rDNA riboprinting beacuse the mt SSU rDNAs do not seem to have introns that are found in the 18S genes of Acanthamoeba and that distort phylogenetic analyses.

• Research in Plant Disease
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• v.11 no.2
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• pp.140-145
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• 2005

A collection of 12 Erwinia carotovora strains from blackleg diseased potato in Jeju island was characterized genetic diversity by 5. cayotovora subsp. atposeptica (Eca)-specific PCR, PCR-RFLP of the two genes (16S rRNA and pel) and repetitive sequence PCR (ERIC-PCR). The results were compared with those of the other E. carotovora representative strains. None of the blackleg strains produced PCR amplicons with Eca-specific primers in contrast to the single 690 bp amplicon obtained with Eca strains. In addition, on the basis of pel gene RFLP with Sau3AI, the blackleg strains belonged to the pattern 2 whereas Eca strains belonged to the other one (pattern 3). By analysis of 16S rDNA RELP generated with HinfI, the most strains including the E. carotovera subsp. carotovora (Ecc) representative strains used in this study belonged to the pattern 1 whereas the blackleg strains belonged to the pattern 2 except for one strain. Moreover, ERIC-PCR analysis showed that the blackleg strains were closely related to each other and had an unique DNA band. Based on these molecular approaches, we have confirmed that the blackleg disease of potato is caused by a different E. carotovora from Eca and Ecc in Jeju island.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.307-312
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• 2004

Diversity of the bacterial communities and the relation between community structure and components of waste­water were analyzed by 16S rRNA-based molecular techniques. Clone libraries of the 16S rDNAs from the sludges were constructed by PCR and cloning. The 1,151 clones from a sludge sample of sewage treatment plant were clustered into 699 RFLP phylotypes and the 1,228 clones from the wastewater disposal plant of chemical industry were clustered into 300 RFLP phylotypes. Shannon-Weiner diversity indices of two sampling sites were 8.7 and 6.1, indicating that the bacterial community structure of sewage treatment plant was more diverse than that of wastewater disposal plant of chemical industry. Forty clones belonging to predominant RFLP types were selected and sequenced. Seventy percent (28 clones) of the sequenced clones were related to the uncultured bacteria in public databases. The ${\beta}-Proteobacteria$ dominated in the bacterial communities of investigated two sludge samples. 16S rDNA sequences of the sewage treatment plant were similar to those of other activated sludges, while the bacterial community in wastewater disposal plant of chemical industry rep­resented the strains identified from high-temperature, anaerobic, hydrocarbon-rich, and sulfur-rich environ­ments. This result suggested that bacterial communities depended upon the components of wastewater.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.815-820
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• 2008

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.366-374
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• 2010

Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Spirastrella abata. A total of 164 bacterial strains associated with the sponge were cultivated using Zobell and Natural sea salt media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 95% similarities compared with known bacterial species, and the isolates belonged to four phyla, Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteriodetes, of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge- derived total gDNA showed five major DGGE bands, and their sequences showed more than 96% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of four phyla, including Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Spirochetes, and Chloroflexi. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with S. abata by both RFLP and DGGE methods; however, overall bacterial community in the sponge differed depending on the analysis methods.

• The Korean Journal of Mycology
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• v.32 no.1
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• pp.1-7
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• 2004

To establish the phylogenetic relationships of Dictyophora spp., rDNA-ITS regions of 11 strains of veiled lady mushroom collected from various countries were amplified and sequenced. It was observed that the 11 strains were divided into four groups based on PCR band patterns of each ITS region cleaved by eight different restriction enzymes in cleaved amplified polymorphic sequence analysis (CAPS). The phylogenic relationship of each group by cleaved amplified polymorphic sequence (CAPS) analysis matches well with previously reported morphological phylogeny, such as 5 strains of D. indusiata, 4 strains of D. echinovolvata, and a strain of Phallus rugulosus. Sequence analysis using the cluster V methods showed more detail classification than CAPS analysis. The 5.8S region showed two point nucleotide base exchanges from G to A according to four groups, and four groups were subdivided by sequence variation of ITS I and ITS II regions. But sequence variation of Phallus rugulosus was not showed in full ITS region. This study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequence, are most useful in systematics and other mushroom study.