• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.148-158
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• 1999

The purpose of this study was to establish a rapid, reliable diagnostic method detecting Encephalomyocarditis virus(EMCV) RNA in formalin-fixed, paraffin-embedded tissues of EMCV naturally infected pigs by cDNA probe of EMC $K_3$, the EMCV strain isolated from Korea. Using a biotin-labelled nick translated probe for the cDNA marker. We made up for some defects of radiolabeled method. In sits hybridization(ISH) technique, differently from the other nucleic acid hybridization methods, is able to detect the virus genome specifically in the state of the intact shapes of cells and/or tissues. We succeeded in performing the experiment to detect the EMCV within 1~2 hours using the $MicroProbe^{TM}$ capaillary action system. In this study, we observed highly specific positive signals of red color by staining the paraffin-embedded tissue sections of naturally EMCV-infected pig organs or tissues, including brain, heart, kidney and lacrimal gland with the Fast Red TR salt/Naphtol phosphate chromogen. The results suggested that this ISH method is considered as a highly sensitive and reliable tool for molecular biologic diagnosis of the EMC viral disease.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.531-536
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• 1995

Salmonella species are the most prevalent etiologic agents of food-borne acute gastroenteritis. Direct isolation of bacteria from the contaminated food, stool and animal tissues has been used for the diagnosis of salmonellosis routinely. However, isolation of bacteria is time consuming work and not so highly sensitive. In recent years, improved methods of polymerization chain reaction(PCR) and probe hybridization technique have led to the developement of diagnostic assays which employ to detect various human and animal pathogenic bacteria. In this study, we have performed the polymerization chain reaction to detect Salmonella pullorum from tissues and stool samples of chickens with two specific primers, ST5 and ST8C. The target DNA fragment of PhoE gene was successfully amplified from liver, spleen, pancreas, heart, lung, ovary, oviduct and feces samples. The amplified DNA fragments were hybridized with Salmonella typhymurium TA3000 PhoE probe by Southern hybridization. The PCR to amplify the PhoE gene was highly rapid and sensitive method to detect Salmonella pullorum from tissues and stool samples.

• Journal of The Korean Society of Grassland and Forage Science
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• v.25 no.4
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• pp.267-274
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• 2005

To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, a thermptolerance gene, DgP23, was introduced into orchardgrass using Agrobacterium - mediated transformation method. PCR and Southern blot analyses using genomic DNA showed specific DNA band on agarose gel and hybridization signal on X- ray film in transgenic orchardgrass harboring the recombinant DgP23 gene, but not in the wild type and empty vector control plants. RT-PCR and Southern blot analyses using total RNA also showed specific DNA band and hybridization signal. Transgenic orchardgrass did not showed ny morphological aberration both in the green house and field cultivation. Thermotolerance of transgenic plants was not detected in laboratory test. but may detected in field test.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.177-182
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• 1993

From 84 clinical isolates of Staphylococcus species, ten strains showing inducible resistance to MLS antibiotics were selected by disk agar diffusion method. Colony hybridization was executed using two MLS inducible resistance genes, ermA and ermC, previously identified from S. aureus as probes. S. hemolyticus 401 and S. epidermidis 542 whose genes were not homologous to those probes were finally selected. It was determined that the resistance genes of S. hemolyticus 401 and S. epidermidis 542 were not homologous to ermA, ermC and ermAM by Southern hybridization. S. epidermidis 542 had a plasmid DNA. To know if the plasmid may have genes related to inducible resistance, it was attempted to transform B. subtilis BR151 and S. aureus RN4220 with the plasmid prepared from S. epidermidis 542. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. hemolyticus 401 and S. epidermidis 542 had novel genes which were not homologous to MLS resistance genes identified previously. It was assumed that these genes may exist in chromosomal DNA.

• Korean Journal of Veterinary Pathology
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• v.1 no.2
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• pp.119-124
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• 1997

The objective of this study was to develop digoxigenin (DIG)-labeled in situ hybridization (ISH) test for diagnosis of Aujeszky's Disease(AD) in infected organs. Specific DNA with well conserved gene sequences encoding gp50 antigen in AD virus (ADV) was obtained by Polymerase Chain Reaction (PCR) method. A pair of oligonucleotide primers used in PCR allowed amplification of a 217 bp sequence from the gp50 ADV gene. The DNA was then labeled with DIG by primer labeling method for use as probe in ISH test to detect ADV nucleic acids in various tissue. Positive hybridization was demonstrated by dark pigmentation in nuclei and cytoplasm of ADV infected cells particularly in brain tonsillar crypt epithelium and pulmonary alveolar cells. This result suggests that ISH is a valuable sensitive and rapid diagnostic test for AD.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.187-193
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• 1996

The T sergenti DNA fragments used as probes of KTS1(2.4kb) and KTS3(1.5kb) were labeled with digoxigenin-11-dUTP for the Southern hybridization. T sergenti DNAs from different geographic locations(Korea; Chonbuk, Kyungbuk, Chungnam, Kangwon, Cheju island, Japan; Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9202, Shintoku 9102) which had been digested with Pst I and EcoR I were probed by the digoxigenin-11-dUTP-labeled KTS1 and KTS3. As the results, the samples from Chonbuk, Kyungbuk, Cheju island in Korea and Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9102 in Japan were positively reacted, but the others from the other locations not reacted. In the comformation test of T sergenti DNA from different geographic locations, all of the samples were positively detected by PCR amplification.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.4
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• pp.379-386
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• 1997

Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.273-278
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• 2000

The aacCl, aacC2, aacC3, and aacC4 genes, which encode aminoglycoside acetyltransferase AAC(3)-I, AA(3)-II, AAC(3)-III, and AAC(3)-IV, respectively, and aerolysin genes in water samples from Juam lake were surveyed by polymerase chain reaction. Surface water was collected from January 1996 to December 1998, and then bacterial DNA was extracted from the water. Twelve samples were tested by PCR to servey the genes for aminoglycoside acetyltransferase and aerolysin in the lake water. The aacC2 gene was detected in 9 of 12 DNA samples. Among 9 samples showing aacC2 positive, 7 samples were associated with Tn3 sequence. However, none of the twelve samples amplified the expected DNA fragment for aacC1, aacC3, and aacC4 genes. PCR primer to detect the aerolysin gene was designed using the conserved region of the genes for aerolysin and hemolysin of Aeromonas spp. This primer set successfully amplified the expected 414 bp PCR product with the DNA samples from the lake water. The aerolysin gene was detected in 7 of 12 DNA samples. When Southern hybridization of the gel with probe was performed, the aerolysin gene was detected in 10 of 12 DNA samples. However, the seasonal fluctuation of these genes was not found.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Korean Journal of Medicinal Crop Science
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• v.26 no.2
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• pp.113-118
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• 2018

Background: Gastrodia elata Blume is a saprophytic perennial plant in the Orchidaceae family, because of its agricultural and medicinal effectiveness, researchers focus on its genome and chemical components. However, cytogenetic information based on the chromosome structure and composition to construct chromosomal backbone for genome sequencing research and for the development and breeding of plants is very limited. Methods and Results: We determined the metaphase chromosome composition of the G. elata genome by fluorescence in situ hybridization (FISH) using 5S and 45S rDNAs and telomeric repeat probes. The nuclear genome of G. elata was organized into 2 n = 36, with relatively small ($2.71-5.50{\mu}m$)chromosomes that showed gradual decrease in size. Conglutination phenomenon was observed among the metaphase chromosomes, and it was distinguished from that in other plant metaphase chromosome spreads. One pair of signal was detected for each 5S and 45S rDNA in the pericentromeric region and interstitial region on the short arm of chromosomes 10 and 4, respectively, and telomeric DNA signals were detected in the terminal region of most chromosomes. Conclusions: To our knowledge, this is the first FISH chromosome composition result in G. elata and could be useful in more comprehensive molecular cytogenetic and genomic analyses as well as breeding programs of the medicinal plant G. elata.