• Korean Journal of Clinical Laboratory Science
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• v.46 no.3
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• pp.99-105
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• 2014

Isolating total DNA from small samples using traditional methods is difficult and inefficient mainly due to loss of DNA during filtration and precipitation. With advances in molecular pathology, DNA extraction from micro-dissected cells has become essential in handling clinical samples. Genomic DNA extraction using small numbers of cells can be very important to successfully PCR amplify DNA from small biopsy specimens. We compared our experimental genomic DNA extraction method (A) with two other commercially available methods: using spin columns (B), and conventional resins (C), and determined the efficacy of DNA extraction from small numbers of cells smeared on a glass slide. Approximately 50, 100, 200, 500 and 1000 cells were isolated from fine needle aspiration biopsy (FNAB) slides aspirated from histologically proven papillary thyroid carcinoma masses. DNA was extracted using the three techniques. After measuring DNA quantity, PCR amplification was performed to detect the ${\beta}$-globin and $BRAF^{V600E}$ gene mutations. DNA extracted by method (A) showed better yield than the other methods in all cell groups. With our method, a suitable amount of genomic DNA to produce amplification was extracted from as few as 50 cells, while more than 100 to 200 cells were required when methods (B) or (C) were applied. Our genomic DNA extraction method provides high quality and improved yields for molecular analysis. It will be especially useful for paucicellular clinical samples which molecular pathologists often confront when handling fine needle aspiration cytology, exfoliative cytology and small biopsy specimens.

• Journal of Practical Agriculture & Fisheries Research
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• v.16 no.1
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• pp.105-113
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• 2014

Equine herpesvirus type 1 (EHV-1) is an economically important pathogen of horses and exerts its major impact by inducing abortion storms and sporadic abortions in pregnant mares, early neonatal death in foals, and respiratory disease in young horses. Although equine herpesvirus type 4 (EHV-4) rarely causes clinical manifestations of disease in organs other than the respiratory track, isolated cases of myeloencephalopathy and sporadic abortions have been reported in EHV-4 infections. Here, we report an abortion storm in Thoroughbred breeding farms in Jeju island, South Korea. It occurred for 16 days from first abortion to last one. There were no clinical signs prior to abortion and stillbirth in broodmares. Two PCR-primers were made on glycoprotein B gene of EHV-1 and EHV-4 to amplify specific common regions of the viruses. We could detect the virus specific genes in aborted samples by PCR, and concluded that the cause of abortion storm was EHV infection. This report describes the first abortion storm case caused by EHV in Thoroughbred breeding farms in South Korea.

• Research in Plant Disease
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• v.15 no.2
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• pp.83-87
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• 2009

Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X axonopodis pv. glycines with DNA concentration as low as 200 fg and $1.8{\times}10^3$ cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

• Journal of Life Science
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• v.27 no.11
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• pp.1331-1339
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• 2017

DNA barcoding is the identification of a species based on the DNA sequence of a fragment of the cytochrome C oxidase subunit I (COI) gene in the mitochondrial genome. It is widely applied to assist with the sustainable development of fishery-product resources and the protection of fish biodiversity. This study attempted to verify horse-head fish (Branchiostegus japonicus) and fake horse-head fish (Branchiostegus albus) species, which are commonly consumed in Korea. For the validation of the two species, a real-time PCR method was developed based on the species' mitochondrial DNA genome. Inter-species variations in mitochondrial DNA were observed in a bioinformatics analysis of the mitochondrial genomic DNA sequences of the two species. Some highly conserved regions and a few other regions were identified in the mitochondrial COI of the species. In order to test whether variations in the sequences were definitive, primers that targeted the varied regions of COI were designed and applied to amplify the DNA using the real-time PCR system. Threshold-cycle (Ct) range results confirmed that the Ct ranges of the real-time PCR were identical to the expected species of origin. Efficiency, specificity and cross-reactivity assays showed statistically significant differences between the average Ct of B. japonicus DNA ($21.85{\pm}3.599$) and the average Ct of B. albus DNA ($33.49{\pm}1.183$) for confirming B. japonicus. The assays also showed statistically significant differences between the average Ct of B. albus DNA ($22.49{\pm}0.908$) and the average Ct of B. japonicus DNA ($33.93{\pm}0.479$) for confirming B. albus. The methodology was validated by using ten commercial samples. The genomic DNA-based molecular technique that used the real-time PCR was a reliable method for the taxonomic classification of animal tissues.

• Development and Reproduction
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• v.4 no.1
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• pp.125-131
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• 2000

This study has used differential display-PCR, to amplify genes transcribed during the ovarian maturation induced by human chorionic gonadotropin (hCG). The cDNA expressed at the times of acquisition of oocyte maturational competence in red seabream (Pagrus major) following treatment with hCG was amplified and cloned. A full-length of cDNA for p. major was isolated using differential display-PCR and 5'RACE. This cDNA clone contained 2,662 nucleotides including the open reading frame that encoded 434 amino acids. Homology analyses, using the GenBank and EMBL general database searches, indicated that the nucleotides sequence of the cDNA does not have high homology with any other genes. This cDNA was judged to be a gene, which induction of maturational competence coincides with increase of mRNA related ovarian maturation. Consensus sequences which were consistent with protein kinase C phosphorylation sites and casein kinase II phosphorylation sites were identified. in vitro, the transcription level of mRNA related ovarian maturation increased between 9hr and 24hr following treatment of ovarian follicles with hCG. It was also increased after GtH-II (300 ng/ml) stimulation. Furthermore, in vivo, mRNA related ovarian maturation was rarely expressed prior to the acquisition of oocyte maturational competence, but was strongly expressed after the acquisition of oocyte maturational competence, suggesting that the hCG induction of maturational competence is brought about by the de novo synthesis of the mRNA related ovarian maturation in p. major.

• Food Science of Animal Resources
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• v.27 no.3
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• pp.351-356
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• 2007

The objective of this study was to develop a rapid and reliable method for the sex determination of beef using the PCR(polymerase chain reaction) technique. We have used two bovine sex determining genes, SRY and ZFY, on the Y-chromosome to identify the sex of Hanwoo and Holstein beet. We attempted to amplify 1,348 bp and 979 bp fragments from male and female genomic DNA corresponding to the SRY and ZFY genes, respectively, using male specific primers. The amplified PCR products were separated by electrophoresis in a 1.5% agarose gel to detect a male specific DNA band. When DNA from male beef was amplified with primers specific for the SRY gene, a DNA band of 1,348 bp was present in all of the male samples, but absent from all of the female samples. Also, when DNA from male beef was amplified with primers specific for the ZFY gene, a DNA band of 979 bp was observed in all of the male samples, but absent from all female samples. In conclusion, the bovine SRY and ZFY genes are typically found only in male beef. For the practical application of this method for the sexing of commercial beef at the processing and marketing stages after slaughter. a total of 350 beef samples collected randomly from local markets were analyzed for sex determination. The proportions of male and female samples were 252 (72%) and 98 (28%), respectively. Therefore. the SRY and ZFY genes. which are specific for the Y-chromosome, may be useful sex-diagnostic DNA markers to distinguish male meat from female meat.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.247-259
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• 1999

Small round structured viruses (SRSV) are the major ethological agents which can cause outbreaks of non-bacterial gastroenteritis or food poisoning both in children and adults. The classification of family Caliciviridae to which SRSV belong, is based on the genome encoding three open reading frames. The rotavirus is another major pathogen which causes diarrhea in young children. We examined stool specimens obtained from diarrheal patients in Wonju from which bacterial pathogens were not found. To detect causative viruses from stool specimens of patients, reverse transcription (RT)-polymerase chain reaction (PCR) or nested PCR using rotavirus or SRSV specific primers was performed. In this study, RT-nested PCR procedure which can amplify a 330 bp fragment derived from RNA dependent RNA polymerase (RDRP) region within ORF1 was applied for the detection of SRSV. For the detection of rotaviruses, a 877 bp fragment from the VP4 region of rotavirus genome was amplified. As a result, rotavirus was not detected while SRSV sequences were detected from one out of five specimens. The nucleotide and amino acid sequences of the Wonju isolate were compared with other 6 Korean isolates which have been isolated and sequenced in our laboratory. Sequence analysis revealed that the Wonju isolate was rather distinct from other Korean isolates: the Wonju isolate was closer to genogroup I of SRSV while other 6 Korean isolates belonged to genogroup II.

• Research in Plant Disease
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• v.14 no.1
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• pp.71-75
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• 2008

An isolate of Peanut stunt virus (PSV), named as Tr-PSV, was isolated from white clover (Trifolium repens L) showing mosaic symptom. Tr-PSV systemically infected all plants tested in the Nicotiana spp. and induced local lesions on inoculated leaves of Chenopodium amaranticolor. However, Tr-PSV induced typical mosaic symptoms as ER-PSV on Vigna unguiculata 5 to 6 days after inoculation, while Fny-CMV used as a control virus of Cucumovirus produced local lesions on inoculated leaves. In dsRNA analysis, Tr-PSV consisted of four dsRNAs, but satellite RNA was not detected. The cDNA of coat protein gene of Tr-PSV was amplified by RT-PCR using a Cucumovirus-specific single pair primers that designed to amplify a DNA fragment of approximately 950 bp. By restriction mapping analysis using RFLP of the RT-PCR products and by serological properties of gel diffusion test, Tr-PSV belongs to a typical member of PSV subgroup I. This is the first report on the occurrence of PSV in white clover in Korea.

• Journal of Veterinary Clinics
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• v.39 no.5
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• pp.207-216
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• 2022

Canine babesiosis has been scarcely investigated in the Republic of Korea (ROK). Although it is known that Babesia gibsoni is its primary causative agent, its clinical presentation has not been completely clarified in the ROK. Consequently, the aim of this study was to evaluate the clinical appearance of this parasitic infection based on the anamnesis of the patient and compare of hematological and biochemical test results. Four hundred whole blood samples from patients with a presumptive diagnosis of tick-borne disease were analyzed by polymerase chain reaction (PCR) to amplify the Babesia spp. 18S rRNA gene and by a rapid diagnostic test kit (VetAll Laboratories^®) to detect B. gibsoni seroreactive animals. Thirty-six (9.0%) dogs were PCR-positive but only 24 (6.0%) were seropositive. The investigation revealed that all the courses of the disease are present in the ROK, with the acute course being predominant. The acute course tends to consist of inappetence, lethargy, pyrexia, gastrointestinal symptoms, and occasionally hematuria. It also occurs with common hematological abnormalities, such as thrombocytopenia and anemia, and to a lesser extent biochemical abnormalities, such as hyperbilirubinemia, hypoalbuminemia, and elevated liver enzymes. This research shows that B. gibsoni is an endemic hemoparasite capable of producing a variety of clinical manifestations in dogs. For its accurate diagnosis, a descriptive history of the clinical signs, hematology, and biochemical profile of the patient, along with a well-performing PCR assay, are essential. These findings will help in planning pragmatic preventive strategies against this potent threat in the ROK.

• Korean Journal of Veterinary Service
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• v.30 no.4
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• pp.505-510
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• 2007

This study was conducted to detect ${\beta}$-lactam antibiotic-resistant genes in the 400 E coli isolates from porcine fecal samples in Korea by a DNA chip. The DNA chip contains the specific probe DNAs of the ${\beta}$-lactam antibiotic-resistant genes that had been labeled with a mixture of primer set designed to amplify specific genes (PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY and AmpC) using a multiplex polymerase chain reaction (PCR). Of 400 isolates 339 contained at least one ${\beta}$-lactamases gene. Resistance to ${\beta}$-lactamases was mediated mainly by AmpC (n = 339, 100%), and followed by TEM (n = 200, 59.0%), CMY (n = 101, 29.8%), PSE (n = 30, 8.9%) and both OXA and SHV genes (n = 20, 5.9%), while the FOX, MEN and OXY genes were not detected. The other sixty-one did not contain any ${\beta}$-lactamase genes even though they were resistant to antimicrobial drugs. In conclusion, the DNA chip system can be used as a rapid and reliable method for detecting of ${\beta}$-lactamases genes, which will help veterinarians select the antibiotics for monitoring and treating of animal diseases.