• Korean Journal of Microbiology
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• v.36 no.3
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• pp.173-180
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• 2000

The intern1 spacer regions (ISR) between the 16s and 23s $1_RNA$ genes of Aeronzonus iwonii blogroupsobria and A. caviae were investigated by PCR fragment length typing and DNA sequencing. A. iwonii bv.sobria has a speciIic 16s-23s pattern of 2-4 fiagments ranging Goin 479-539 bp, with the exception of thespecies Aeron7onns cmiae, which has 3 fragments ranglog from 470-602 bp. In all of the.4 vei*onii bv. sobr,iaand A, caviae strains examined in this study, the 470-481bp Tragnent, designated TSR-1, invariably contained $tDNA^{uc(GAT)$ and $tDNA^{Ala(TGC)$ in contrast to ISR-2 (513-525 bp). ISR-3 (537-539 bp) and ISR-4 (568-602 bp)containing TEX>$tDNA^{Olu(ITC)$ A stretch of 20 nucleotides (178-197 bp) in the ISR-4 was conserved only wit11mA.caiiue, from which the A. caiiae specific primer, named prAC-F, was designed and used for PCR with aAcaviae coimnon reverse primer A PCR product of 450 bp was apparent alnong I , caiizne strains, but not ii1.4.ijeronii bv. sob~ia strains. The PCR product was oot detected t"-om strains belonging to A. hjili-o~~hila, Ebrio,aud the family Ef\ulcornertei,obncteriaceae. This study provides the first molecular tool for mdentifying the species 8.caviae.ing the species 8. caviae.

• Proceedings of the Korean Aquaculture Society Conference
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• 2004.05a
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• pp.240-241
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• 2004

• Journal of fish pathology
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• v.17 no.3
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• pp.159-169
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• 2004

Bacterial wihte enteritis ocurred by infection of V. ichthyoenteri is a devastating disease in olive flounder (Paralichthys olivaceus) hatcheries in Korea. Since white enteritis has been a problem in aquqtic industries, necessity of a rapid detection method is increased. In an attempt to develop rapid PCR method the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. The intergenic spacers were amplified by primers complementary to conserved region of 16S and 23S rRNA genes. The intergenic spacer region between the 16S and 23S rRNA genes of V. ichthoenteri were investigated by PCR fragment length typing and DNA sequencing. Analysis of the ISR sequences showed that V. ichthyoenteri contains one types of polymorphic ISRs. The size of ISRs ranged 348bp length and not contains tRNA genes. Mutiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of Vibrio ichthyoenteri. PCR. The specific of the primer was examined using genomic DNA prepared from 19 different Vibrio species, isolated 18group Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1046-1054
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• 2001

Bioleaching is the process in which insoluble metal sulfide is oxidized by specialized iron- and/or sulfur-oxidizing lithotrophic bacteria in acidic, metal-rich environments. Most of these processes are carried out by the genus Thiobacillus. Three novel Thiobacillus strains (Thiobacillus thiooxidans AZ11, Thiobacillus thiooxidans MET, and thiobacillus thiooxidans TAS) associated with bioleaching have been isolated from soil and sludge (Korean patent No. 1999-0073060 for T. thiooxidans AZ11, Korean patent No. 1999-0005798 for T. thiooxidans MET, and Korean patent No. 1999-0073059 for T. thiooxidans TAS). A partial sequence of 16S ribosomal RNA gene (16S rDNA) and the entire sequence of 16S/23S intergenic spacer region (ISR) were determined in the three above novel strains and in Thiobacillus ferrooxidans ATCC19859 as a reference strain. When phylogenetic analysis was performed based on G+C contents and sequence alignments, T. ferroxidans ATCC19859 was found to be closely related to previously registered T. ferrooxidans strains in a monophyletic manner, while the three novel T. thiooxidans strains were classified in a paraphyletic manner. Close examination on the base composition of 16S/23S ISR revealed that the 5\` part (nucleotide residues 21-200) was specific for the genus Thiobacillus. On the other end, the 3\` part (nucleotide residues 201-520) showed specificity in T. ferrooxidans strains, but not in T. thiooxidans strains. These results suggest that the proximal and distal halves of 16S/23S could be used as a genetic marker for the identification of the genus Thiobacillus and the species T. ferrooxidans, respectively.

• Korean Journal Plant Pathology
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• v.14 no.4
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• pp.350-357
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• 1998

A total of 12 strains of fluorescent Pseudomonas isolated from the cultivated mushrooms such as Agaricus bisporus and Pleurotus ostreatus were collected. They consisted of pathogenic Pseudomonas spp. and epiphytic Pseudomonas spp. of the cultivated mushroom. To analyze the phylogenetic relationship of these strains, ITS I region, the 16S-23S intergenic spacer region in the ribosomal RNA (rRNA) operon, was cloned and sequenced. The spacer regions of these strains were 495∼527 nucleotides in length and contained the genes encoding isoleucine-tRNA (tRNAIle) and alanine-tRNA (tRNAAla). The reciprocal homologies of each ITS I sequence among these strains were in the range of 84.2%∼98.8%. According to the analysis of ITS I sequences, the fluorescent Pseudomonas spp. were phylogenetically classified into three clusters. Cluster I consisted of Pseudomonas fluorescens, P. tolaasii, P. gingeri’, and P.‘reactans’(WLRO). Cluster II comprised Pseudomonas fluorescens biovar C and F. Cluster III composed P. agarici. Cluster I and II could be classified into P. fluorescens complex. P. agarici formed an independent taxon clearly separable from P. florescens complex.

• Journal of Life Science
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• v.28 no.9
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• pp.1118-1126
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• 2018

In this study, investigated the general characteristics and diagnostic methods types of streptococcosis among various fish disease pathogens that caused a lot of economic damaged to aquaculture fish based on the previous research paper. Streptococcosis infection of fish is considered a reemerging disease affecting a variety of wild and cultured fish throughout the world. Calssifiacation of Gram positive cocci based on DNA-DNA hybridization coupled with 16S sequencing has shown that at least five different species are considered of significance as fish pathogens: Lactococcus garvieae, L. piscium, Streptococcus iniae, S. agalactiae, S. paruberis, Vagococcus salmoninarum. Symptoms of infection with streptococcosis disease such as body color change, eyeball abnormality, gill discoloration, bleeding, abdominal distension, swelling of the kidney and spleen. In addition, it usually occurs from June to October when the water temperature rise a lot of fish death. Currently, 16S rRNA, 16S-23S rRNA intergenic spacer region (ISR), Random Amplified polymorphic DNA (RAPD), Ribotyion (RT), Loop-mediated isothermal amplification (LAMP) are among the methods for diagnosing streptococcosis. Among them, the LAMP method, which is high applicable to the aquaculture farm has attracted the spotlight, but due to problems such as confirmation of results. This seems to minimize the economic loss of streptococcosis which complements the problem so that it can be easily used from the diagnosis to the results confirmation.

• Microbiology and Biotechnology Letters
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• v.48 no.2
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• pp.193-204
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• 2020

Sixteen acetic acid bacteria (AAB) were isolated from blueberries and citric fruits of the Salto Grande region (Concordia, Entre Rios, Argentina) using enrichment techniques and plate isolation. Enrichment broths containing ethanol and acetic acid enabled maximum AAB recovery, since these components promote their growth. Biochemical tests allowed classification of the bacteria at genus level. PCR-RFLP of the 16S rRNA and PCR-RFLP of the 16S-23S rRNA intergenic spacer allowed further classification at the species level; this required treatment of the amplified products of 16S and 16S-23S ITS ribosomal genes with the following restriction enzymes: AluI, RsaI, HaeIII, MspI, TaqI, CfoI, and Tru9I. C7, C8, A80, A160, and A180 isolates were identified as Gluconobacter frateurii; C1, C2, C3, C4, C5, C6, A70, and A210 isolates as Acetobacter pasteurianus; A50 and A140 isolates as Acetobacter tropicalis; and C9 isolate as Acetobacter syzygii. The bacteria identified by 16S rRNA PCR-RFLP were validated by 16S-23S PCR-RFLP; however, the C1 isolate showed different restriction patterns during identification and validation. Partial sequencing of the 16S gene resolved the discrepancy.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.490-495
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• 2007

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.

• Journal of fish pathology
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• v.19 no.2
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• pp.127-139
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• 2006

From 2002 to 2004, various vibrios were isolated from the olive flounder, Paralichthys olivaceus of culturing size with disease signs. During this survey, it was known that the high proportion of Vibrio ichthyoenteri was occupied among the isolated vibrios. Generally, V. ichthyoenteri is well known as the pathogen of bacterial enteritis of olive flounder larvae. The aim of the present study was the compare the characteristics of two groups of V. ichthyoenteri, culturing sized olive flounder, and larvae of olive flounder showing the intestinal necrosis. The research was focused on the physiology, biochemistry, genetics in the two bacterial groups. The physiological and biochemical characteristics of the tested strains were very similar. The intergenic spacer (IGS) region between the 16S and 23S rRNA genes of 21 isolated strains and 3 reference strains, V. ichthyoenteri, were investigated by PCR fragment length typing and DNA sequencing. After the isolated strains were identified as V. ichthyoenteri, not only phenotypic characteristics of the isolated and reference strains but also homology of 16S-23S IGS of all isolated strains and reference strains as 99.1~100%. The V. ichthyoenteri showed 4 specific 16S-23S patterns and contained no-tRNA, tRNAGlu(TTC) , tRNAIle(GAT) tRNAAla(TGC) type .

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.321-327
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• 2015

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64℃. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10³ CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.