• Journal of Microbiology and Biotechnology
• /
• v.20 no.8
• /
• pp.1163-1178
• /
• 2010

Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of seven Vibrio plasmid replicons. Comparative sequences analysis of the seven Vibrio plasmid replicons obtained in this study together with eight published Vibrionaceae plasmid sequences revealed replication-participating elements involved in the ColE1 mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation, and as a result, the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids, based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids were demonstrated by direct sequencing analysis and Southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNA I species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing a basis for the rapid characterization and classification of Vibrionaceae plasmids.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.51 no.3
• /
• pp.248-253
• /
• 2018

Freshwater farms are primarily located adjacent to rivers and lakes, facilitating the introduction and spread of pathogens into natural systems. Therefore, it is necessary to continuously monitor natural aquatic organisms, the breeding environment, and infection rates by pathogenic organisms. Fish and crustaceans were sampled 4 times in the Geum River estuary in 2016. The samples were analyzed for the presence of pathogens for reportable communicable diseases, including KHVD (koi herpesvirus disease), SVC (spring viraemia of carp), EUS (epizootic ulcerative syndrome) and WSD (white spot disease); parasite abundance was also examined. The dominant fish species were deep body bitterling Acanthorhodes macropterus (21.4%), followed by skygager Erythroculter erythropterus (12.7%). For crustaceans, Palaemon paucidens and Chinese mitten crab Eriocheir sinensis were dominant. Sixty fish and 36 crustacean species were examined for reportable communicable diseases. When using a specific primer set for each disease, PCR analysis did not detect any reportable communicable diseases in the samples. Some instances of Dactylogyrus, copepods, nematodes and metacercaria were detected. However, the PCR results indicated that the metacercaria were not Clonorchis sinensis.

• Microbiology and Biotechnology Letters
• /
• v.38 no.4
• /
• pp.455-460
• /
• 2010

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis was applied to detect and identify ten Weissella spp. frequently found in kimchi. The previously reported genus-specific primers designed from 16S rDNA sequences of Weissella spp. were adopted but PCR was performed at the increased annealing temperature by $4^{\circ}C$. The sizes of amplified PCR products and restricted fragments produced by AluI, MseI, and BceAI endonucleases were well correspond with the expected sizes. W. kandleri, W. koreensis, W. confusa, W. minor, W. viridescens, W. cibaria, and W. soli were distinguished by AluI and MseI and W. hellenica and W. paramesenteroides were identified by BceAI. W. thailandensis was distinguished when restriction pattern of other species was compared but identified by the single use of MspI.

• Biomedical Science Letters
• /
• v.25 no.4
• /
• pp.407-416
• /
• 2019

Recent years have seen an increase in the incidence of candidiasis caused by non-albicans Candida (NAC) species. In fact, C. glabrata is now second only to C. albicans as the most common cause of invasive candidiasis. Therefore, the rapid genotyping specifically for C. glabrata is required for early diagnosis and treatment of candidiasis. A number of genotyping assays have been developed to differentiate C. glabrata sequence types (STs), but they have several limitations. In the previous study, multi-locus sequence typing (MLST) has performed with a total of 101 C. glabrata clinical isolates to analyze the prevalent C. glabrata STs in Korea. A total of 11 different C. glabrata STs were identified and, among them, ST-138 was the most commonly classified. Thus, a novel probe-based quantitative PCR (qPCR) assay was developed and evaluated for rapid and accurate identification of the predominant C. glabrata ST-138 in Korea. Two primer pairs and hybridization probe sets were designed for the amplification of internal transcribed spacer 1 (ITS1) region and TRP1 gene. Analytical sensitivity of the probe-based qPCR assay was 100 ng to 10 pg and 100 ng to 100 pg (per 1 μL), which target ITS1 region and TRP1 gene, respectively. This assay did not react with any other Candida species and bacteria except C. glabrata. Of the 101 clinical isolates, 99 cases (98%) were concordant with MLST results. This novel probe-based qPCR assay proved to be rapid, sensitive, highly specific, reproducible, and cost-effective than other genotyping assay for C. glabrata ST-138 identification.

• Mycobiology
• /
• v.48 no.4
• /
• pp.313-320
• /
• 2020

In Pleurotus sp., green mold, which is considered a major epidemic, is caused by several Trichoderma species. To develop a rapid molecular marker specific for Trichoderma spp. that potentially cause green mold, eleven Trichoderma species were collected from mushroom farms and the Korean Agricultural Culture Collection (KACC). A dominant fungal isolate from a green mold-infected substrate was identified as Trichoderma pleuroticola based on the sequences of its internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) genes. In artificial inoculation tests, all Trichoderma spp., including T. atroviride, T. cf. virens, T. citrinoviride, T. harzianum, T. koningii, T. longibrachiatum, T. pleurotum, and T. pleuroticola, showed pathogenicity to some extent, and the observed symptoms were soaked mycelia with a red-brown pigment and retarded mycelium regeneration. A molecular marker was developed for the rapid detection of wide range of Trichoderma spp. based on the DNA sequence alignment of the ITS1 and ITS2 regions of Trichoderma spp. The developed primer set detected only Trichoderma spp., and no cross reactivity with edible mushrooms was observed. The detection limits for the PCR assay of T. harzianum (KACC40558), T. pleurotum (KACC44537), and T. pleuroticola (CAF-TP3) were found to be 500, 50, and 5 fg, respectively, and the detection limit for the pathogen-to-host ratio was approximately 1:10,000 (wt/wt).

• Animal Bioscience
• /
• v.34 no.4
• /
• pp.734-742
• /
• 2021

Objective: The present study aimed to investigate the occurrence and species of coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS) in retail pork meat samples collected during nationwide monitoring. The staphylococcal isolates were characterized for antimicrobial and zinc chloride resistance and enterotoxigenic potential. Methods: A total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea for isolation of staphylococci. Antimicrobial and zinc chloride resistance phenotypes, and genes associated with the resistance phenotypes were determined on the isolates. Furthermore, the presence and distribution of 19 staphylococcal enterotoxin (SE) genes and enterotoxin-like genes among the pork-associated staphylococci were determined by multiplex polymerase chain reaction-based assays using the specific primer sets. Results: A total of 29 staphylococcal strains (29/260, 11.1%) were isolated from samples of retail pork meat, 24 (83%) of which were CoNS. The four CoNS species identified were S. saprophyticus (n = 16, 55%), S. sciuri (n = 3, 10%), S. warneri (n = 3, 10%), and S. epidermidis (n = 2, 7%). Among the 29 isolates, four methicillin-resistant CoNS (MR-CoNS; three S. sciuri and one S. epidermidis) and one methicillin-resistant CoPS (MR-CoPS; one S. aureus) were identified. In addition, a relatively high level of tetracycline (TET) resistance (52%) was confirmed in CoNS, along with a predominant distribution of tet(K). The most prevalent SEs were sep (45%), and sen (28%), which were carried by 81% of S. saprophyticus. Conclusion: These findings suggest that CoNS, especially S. saprophyticus strains, in raw pork meat could be a potential risk factor for staphylococcal food poisoning (SFP), and therefore, requires further investigation to elucidate the role of SEls in SFP and virulence of the pathogen. Our results also suggest that CoNS from raw pork meat may act as a source for transmission of antimicrobial resistance genes such as staphylococcal cassette chromosome mec and tet(K).

• Applied Biological Chemistry
• /
• v.47 no.2
• /
• pp.182-186
• /
• 2004

Morphological observation of roots and molecular technique were used to investigate the symbiotic relationships between arbuscular mycorrhizal (AM) fungi and ginseng roots. Korean ginseng, Panax ginseng, was collected from 8 sites in Korea. Colonization pattern of AM fungi in ginseng roots was determined as an Arum type under light microscopes. Nested PCR using AM fungal specific primers was employed to amplify a partial region on 18s rDNA of AM fungi from the root extracted mixed DNA. The amplified DNA was cloned and analyzed by random fragment length polymorphism (RFLP) with restriction enzymes, AluI, HinfI and AsuC21. One from each RFLP pattern was selected for sequencing. A total 16 clones were sequenced and identified as 2 species of AM fungi; Paraglomus brasilianum and Glomus spurcum. Paramglomus brasilianum was found from most of the ginseng roots, in this syudy suggesting that this species of AM fungi could have specific relationship with the ginseng root. Possible roles of AM fungal species in ginseng roots are discussed.

• Development and Reproduction
• /
• v.14 no.4
• /
• pp.261-268
• /
• 2010

As in the O. mykiss electrophoretic profiles of RNA, the signals of each RNA sample from 9 individual tissues such as liver, muscle, brain, heart, pituitary gland, kidney, intestine, spleen and gill similar to positive control were obtained. The tissue distributions of the complimentary DNA (cDNA) of O. mykiss four genes were analyzed using quantitative real-time PCR with primer sets for tissue expression analysis. In this rainbow trout species, author obtained bands of various sizes, ranged from 700 bp to 1,400 bp. A dissociation curve was made at the end of each run to make sure that there was no non-specific amplification. Supplementarily, the Ct of each DNA was compared. The Ct values of vinculin with rainbow trout tissues were determined in a manner similar to those for agouti-related protein (AgRP) and melanocortin receptors (MC4R I and MC4R II). Further, obtained Cts for standard curve of each DNA were affected by specific product (vinculin, AgRP and MC4R II genes). After several experiments with four individual genes of rainbow trout, author estimated a variation ratio of the mean Ct value of the DNA extracted using the comparative CTt method was 37.27, and the standard deviation was 5.33. The correlation coefficient between the Ct values and the concentration of cDNA was -0.98, -0.99, -0.91 and -0.86, respectively (vinculin, AgRP, MC4R I and MC4R II genes). Since this correlation showed high linearity, the straight line obtained was used as a standard for the O. mykiss tissues reared in aquarium. A PCR efficiency of 100% is ideally achieved when the slopes are close to the theoretical value of -3.31. According to quantification method, the results of quantification are strongly affected by the DNA fragmentation. The size of most DNA fragments obtained from various tissues of rainbow trout used in the experiment was approximately 100 bp. According to the four slopes, an efficiency of nearly 100% was estimated for four genes detection methods. Additionally, further analysis with more individuals and primers will be required to fully establish optimization in rainbow trout.

• The Korean Journal of Malacology
• /
• v.24 no.1
• /
• pp.11-24
• /
• 2008

GDNA was isolated from the jedo venus clam (Protothaca jedoensis, Lischke) from Boryeong (jedo venus clam from Boryeong JVCB) and Wonsan (jedo venus clam from Wonsan; JVCW) located in the West Sea and the East Sea of Korean Peninsula, respectively and we performed clustering analyses, DNA polymorphisms and the populations genetic variations. In the present study, the seven decamer primer generated the one hundred and eleven major/minor specific bands in JVCB population and ninety four-specific bands in JVCW population. Seven primers generated the unique shared bands to each population of one hundred and seventy-six, on average of 25,1, in JVCB population from Boryeong and three hundred thirty, on average of 47,1, in JVCW population from Wonsan, respectively. The dendrogram obtained by the seven oligonucleotides primers, indicates two genetic clusters. Especially, two Protothaca between the individual WONSAN no. 12 and BORYEONG no. 10 showed the longest genetic distance (0.537) in comparison with other individuals used. Accordingly, RAPD analysis showed that the JVCB was a little more genetically diverse than the JVCW population. This result implies the genetic similarity owing to rearing in the same and/or similar circumstances or inbreeding within the JVCW population. So to speak, JVCB population may have high levels of genomic DNA variability owing to the introduction of the wild individuals from the other sites to sampling sites although it may be the geographically diverse distribution of this species. However, it was confirmed that it did not appear like that really in this study. We feel convinced that RAPD analysis discovered a significant genetic distance between two Protothaca population pairs (P<0.001). The existence of population discrimination and genetic diversity between two Protothaca populations was identified by RAPD analysis.

• Research in Plant Disease
• /
• v.15 no.2
• /
• pp.88-93
• /
• 2009

In 2008, leaf rot and blight on pepper seeding ("Dokya-chungchung") occurred in a pepper farm at Hwaseong-si, Gyeonggi-do, Korea. The typical symptom is water-soaking and dark brown leaf blight at edges and tips of leaves. The fungal colonies isolated from infected tissues were pinkish at first and turned gradually to gray. Conidia were fusiform, non-septum, and $8.1-17.0{\times}2.0-3.8{\mu}m$ in size. Several specific PCR primers derived from the sequence of the internal transcribed spacer (ITS) region of the rDNA, such as CaINT, CgINT and CcINT were used for the identification of the fungal pathogen. The C. acutatum-specific primer CaINT was amplified single fragment of 496 bp that discriminated C. acutatum from the other species. The pathogenicity test was performed on seedlings and fruits of red pepper. On the basis of the morphological, molecular characteristics and pathogenicity test, we identified as Colletotrichum acutatum. This is the first report on leaf rot and blight on pepper seedling caused by C. acutatum in Korea.