• Journal of Yeungnam Medical Science
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• v.12 no.2
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• pp.366-385
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• 1995

Epidemiological studies are important in both the prevention and treatment of mycobacterial infections. This study was initiated to establish the pulsed-field gel electrophoresis (PFGE) method, which are not yet extensively studied. The most apprpriate restriction endonucleases included DraI, AsnI, and XbaI. The optimal PFGE condition was different according to the enzymes used. Two stage PFGE was performed, in case of DraI first stage was performed with 10 seconds of initial pulse and 15 seconds of final pulse, while the second stage was performed with 60 seconds of initial pulse and 70 seconds of final pulse. The electrophoresis time for DraI-PFGE was 14 hours for each stage. Electrophoresis was performed for 22 hours, in case of XbaI, with 3 seconds of initial pulse and 12 seconds of final pulse. Electrophoresis was performed for 22 hours, in case of AsnI, with 5 seconds of initial pulse and 25 seconds of final pulse. In all cases the voltage of the electrophoresis was maintained constantly at 200 voltage. Standard mycobacterial strains, which included Mycobacterium bovis BCG, M. tuberculosis, and M. fortuitum, could not be differentiated by PFGE analysis. PFGE analysis was performed to differentiate 9 clinically isolated M. fortuitum strains using AsnI. All M. fortuitum strains showed different genotypes except 2 strains. Cluster analysis divided M. fortuitum strains into 2 large groups. PFGE analysis was performed to further differentiate M. fortuitum isolates using XbaI. The undifferentiated 2 M. fortuitum strains showed different PFGE patterns with Xba I. Cluster analysis of the XbaI-PFGE patterns showed more complex grouping than AsnI-PFGE patterns, which showed that XbaI-PFGE analysis was better than AsnI-PFGE in M. fortuitum genotyping. The top dissimilarity values of AsnI-PFGE and XbaI-PFGE were 0.74 and 0.75, respectively. This value was higher than that of arbitrarily primed polymerase chain reaction (AP-PCR) analysis and lower than that of restriction fragment length polymorphism (RFLP) analysis. This suggested that PFGE can be used as a supportive or alternative genotyping method to RFLP analysis.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.367-374
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• 1997

Bovine rotaviruses(A, 288, 55086 strains) isolated from fecal samples in Korea were propagated onto MA104 cells and were confirmed tentatively as G6, G8, and G10, respectively, by RFLP analysis. Full-length VP7 gene of these isolates was amplified by reverse transcriptase polymerase chain reaction(RT-PCR) using VP7 specific primers and cloned into TA vector. Nucleotide and deduced amino acid sequences of VP7 genes of the isolates were determined and compared with those of bovine rotavirus reference strains(NCDV; G6, UK; G6, Cody I-801; G8 and B223; G10). A, 288 and 55086 isolates showed high degree of nucleotide sequence homology with NCDV and UK(93% and 94%), Cody I-801(86%) and B223(97%), respectively, However, they showed 71~74% of nucleotide sequence homlogy with bovine rotavirus reference strains which belong to different serotypes. From the results of deduced amino acid sequence homology analysis, three isolates showed 94~96% of homology with the same serotype reference strains but 80~84% of homology with the different serotype reference strains. Three bovine rotavirus isolates, A, 288 and 55086 strains, were confirmed as G6, G8, and G10, respectively, by nucleotide and deduced amino acid sequence analysis.

• The Korean Journal of Mycology
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• v.25 no.1 s.80
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• pp.46-67
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• 1997

From 1985 to 1993, we collected 20 isolates throughout Kangwon and obtained 6 isolates from other sources. A. mellea formed rhizomorph actively, and some of A. osroyae were poor in the formation of rhizomorph and some without formation of rhizomorph. A. tabescens was active in the growth of aerial mycelium and poor in the development of rhizomorph. In A. gallica, the mycelium development among the isolates were variable greatly, and especially in isolate A8(KNU-250), the mycelial development was similar to that of A. osroyae, but A8(KNU-250) showed the feature of A. gallica to change medium into brown color. In PCR-RFLP analysis of the IGS region in rDNA, the homology between each isolate in the A. mellea and A. ostoyae showed 100% homology. A. tabescens showed $0.919{\sim}0.974$ homology, and A. gallica showed $0.619{\sim}1.000$ homology. A19 and A12 showed 100% homology as the same group, but compared with other subgroups they showed less than 10% homology as $0.051{\sim}0.108$ value. In RAPD analyses, the isolates of A. mellea showed high homology among themselves as $0.983{\sim}1.000$, and A. ostoyae also showed high similarity. The homology between isolates of A. tabescens showed $0.594{\sim}0.953$ value because A. gallica showed $0.280{\sim}0.733$ value, and the variations between isolates were greater than other species. Especially, A19 and A22 were identified as new novel group which were remoted from other groups, and the homology between these two isolates showed 0.921 value, and the genetic similarity between these groups and other 4 groups showed less than 7% as $0.012{\sim}0.069$ value. Of 5 species identified in this study, 4 species were identified as A. mellea, A. ostoyae, A. tabescens, and A. gallica that were already reported ones and 1 species was suggested as a new specie in Korea.

• Korean journal of applied entomology
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• v.53 no.1
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• pp.85-91
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• 2014

In this study, we analyzed the phylogenetic characterization of root-knot nematodes (Meloidogyne spp.) in soils from fruits and vegetables greenhouses in Korea. Soil samples were collected from 12 greenhouse fields in which tomato, cucumber, watermelon, and Oriental melon were being cultivated. Meloidogyne spp. were detected in all the soil samples at an average number of $72{\pm}6$ nematodes/300 g of soil to $2,898{\pm}468$ nematodes/300 g of soil. Phylogenetic analysis using polymerase chain reaction-restriction fragment length polymorphism was attempted for the second-stage juveniles (J2) of Meloidogyne spp. collected from the greenhouse soils. Twelve Meloidogyne spp. from the greenhouse soils were classified into two groups by using HinfI digestion of mitochondrial DNA, resulting in 900, 410, 290, and 170 bp fragments (group A) and 900, 700, and 170 bp fragments (group B). Phylogenetic analysis based on mitochondrial DNA sequences (1,483-1,521 bp) showed that nine group A isolates were identified as Meloidogyne incognita (99.73-99.93%) and three group B isolates showed 99.54-99.73% similarity to Meloidogyne arenaria.

• Korean Journal of Microbiology
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• v.37 no.2
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• pp.137-144
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• 2001

In order to investigate the diversity of bacterial community in the mud flat of Sunchon Bay, Chunnam province, diversity of amplified 16S rDNA was examined. Total DNA was extracted from sediment soils and 16S rDNAs were amplified using PCR primers based on the universally conserved sequences in bacteria. Clonal libraries were constructed and 111 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII. Clones were clustered based on restriction patterns using computer program, GelCompar II. One hundred different RFLP types were detected from 111 clones. The 20 clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the bacterial diversity in the clone libraries. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA databases and GenBank. All sequences showed between 77 and 96.8% similarity to the known 16s rRNA sequence from cultured organisms. The 20 clones sequenced fell into seven major lineages of the domain Bacteria: alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga) and Cyanobacteria (chloroplast). Among the clones, the Proteobacteria were dominant.

• Mycobiology
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• v.31 no.2
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• pp.68-73
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• 2003

Roots of Glycine max and Miscanthus sinensis and soil samples were collected from various field sites at Goesan, Chungbuk in Korea. Microscopic observations of the roots indicated high colonization rates of both arbuscular mycorrhizal fungi(AMF) and other fungi. The partial small subunit of ribosomal DNA genes were amplified with the genomic DNA extracted from their roots by nested polymerase chain reaction(PCR) with universal primer NS1 and fungal specific primers AML Restriction fragment length polymorphism(RFLP) was analyzed using the combinations of three restriction enzymes, HinfI, AluI and AsuC21. Nucleotides sequence analysis revealed that ten sequences from Miscanthus sinensis and one sequence from Glycine max were close to those of arbuscular mycorrhizal fungi. Also, 33% of total clones amplified with NS31-AM1 primers from M. sinensis and 97% from G. max were close to Fusarium oxysporum or other pathogenic fungi, and they were successfully distinguished from AME Results suggested that these techniques could help to distinguish arbuscular mycorrhizal fungi from root pathogenic fungi in the plant roots. Especially, DNA amplified by these primers showed distinct polymorphisms between AMF and plant pathogenic species of Fusarium when digested with AsuC21.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1701-1707
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• 2005

This study deals with the characterization of porcine PIK3C3 and association tests with quantitative traits. PIK3C3 belongs to the class 3 PI3Ks that participate in the regulation of hepatic glucose output, glycogen synthase, and antilipolysis in typical insulin target cells such as those in the such as liver, muscle system, and fat. On the analysis of full-length mRNA sequence, the length of the PIK3C3 CDS was recorded as 2,664 bps. As well, nucleotide and amino acid identities between human and pig subjects were 92% and 99%, respectively. Five SNPs were detected over 5 exons. We performed genotyping by using a SNP C2604T on exon24 for 145 F$_2$ animals (from a cross between Korean native boars and Landrace sows) by PCR-RFLP analysis with Hpy8I used to investigate the relationship between growth and fat depot traits. In the total association analysis, which doesn' consider transmission disequilibrium, the SNP showed a significant effect (p<0.05) on body weight and carcass fat at 30 weeks of age as well as a highly significant effect (p<0.01) on back fat. In an additional sib-pair analysis, C allele still showed positive and significant effects (p<0.05) on back fat thickness and carcass fat. Moreover, the effects of C allele on the means of within-family components for carcass fat and back fat were estimated as 2.76 kg and 5.07 mm, respectively. As a result, the SNP of porcine PIK3C3 discovered in this study could be utilized as a possible genetic marker for the selection of pigs that possess low levels of back fat and carcass fat at the slaughter weight.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.15-20
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• 2016

Toxoplasmosis is an important zoonotic disease that can cause abortion in humans and animals. The aim of this study was isolation and subsequent genotyping of Toxoplasma gondii isolates in ovine aborted fetuses. During 2012-2013, 39 ovine aborted fetuses were collected from sheep flocks in Khorasan Razavi Province, Iran. The brain samples were screened for detection of the parasite DNA by nested PCR. The positive brain samples were bioassayed in Webster Swiss mice. The serum samples of mice were examined for T. gondii antibodies by IFAT at 6 weeks post inoculation, and T. gondii cysts were searched in brain tissue samples of seropositive mice. The positive samples were genotyped by using a PCR-RLFP method. Subsequently, GRA6 sequences of isolates were analyzed using a phylogenetic method. The results revealed that T. gondii DNA was detected in 54% (20/37, 95% CI 38.4-69.0%) brain samples of ovine aborted fetuses. In bioassay of mice, only 2 samples were virulent and the mice were killed at 30 days post inoculation, while the others were non-virulent to mice. The size of cysts ranged $7-22{\mu}m$. Complete genotyping data for GRA6 locus were observed in 5 of the 20 samples. PCR-RLFP results and phylogenetic analysis revealed that all of the isolated samples were closely related to type I. For the first time, we could genotype and report T. gondii isolates from ovine aborted fetuses in Khorasan Razavi Province, Iran. The results indicate that the T. gondii isolates are genetically related to type I, although most of them were non-virulent for mice.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.423-429
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• 2016

Cryptosporidium is an important pathogen causing gastrointestinal disease in snakes and is distributed worldwide. The main objectives of this study were to detect and identify Cryptosporidium species in captive snakes from exotic pet shops and snake farms in Thailand. In total, 165 fecal samples were examined from 8 snake species, boa constrictor (Boa constrictor constrictor), corn snake (Elaphe guttata), ball python (Python regius), milk snake (Lampropeltis triangulum), king snake (Lampropeltis getula), rock python (Python sebae), rainbow boa (Epicrates cenchria), and carpet python (Morelia spilota). Cryptosporidium oocysts were examined using the dimethyl sulfoxide (DMSO)-modified acid-fast staining and a molecular method based on nested-PCR, PCR-RFLP analysis, and sequencing amplification of the SSU rRNA gene. DMSO-modified acid-fast staining revealed the presence of Cryptosporidium oocysts in 12 out of 165 (7.3%) samples, whereas PCR produced positive results in 40 (24.2%) samples. Molecular characterization indicated the presence of Cryptosporidium parvum (mouse genotype) as the most common species in 24 samples (60%) from 5 species of snake followed by Cryptosporidium serpentis in 9 samples (22.5%) from 2 species of snake and Cryptosporidium muris in 3 samples (7.5%) from P. regius.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.165-170
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• 2006

Using the somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel, porcine SOCS2 gene was mapped at SSC5 (1/2) q21-q24 and closely linked with SW1383 marker (47 cR in distance), while SOCS3 gene was assigned to SSC12p11-(2/3p13) and closely linked with SW2490 (43 cR). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the expression of these two genes in the different tissues and the results showed that both SOCS2 and SOCS3 genes were widely expressed in tissues investigated (heart, liver, spleen, lung, kidney skeletal muscle, fat and brain), although some tissues showed lower gene expression. Moreover, SOCS2 and SOCS3 genes had different expression levels at different stages, in different tissues and in different breeds. A G/A substitution, which can be recognized by restriction enzyme of Cfr421, was observed in 5' untranslated region (5'-UTR) of SOCS2 gene. The allele frequencies was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) method and it showed that the allele frequency among Dahuabai, Erhualian, Yushan, Qingping, Large white and Landrace tested were different. Association analysis in a cross experimental populations revealed no significant association between the SOCS2 gene polymorphism and the economic traits investigated. The full-length coding regions (CDs) of porcine SOCS3 gene was obtained by RT-PCR.