• Journal of Embryo Transfer
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• v.28 no.4
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• pp.355-360
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• 2013

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.6
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• pp.888-895
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• 2015

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and subsequent sub-cloning and sequencing were used in this study to analyze the molecular phylogenetic diversity and spatial distribution of bacterial communities in different spatial locations during the cooling stage of composted swine manure. Total microbial DNA was extracted, and bacterial near full-length 16S rRNA genes were subsequently amplified, cloned, RFLP-screened, and sequenced. A total of 420 positive clones were classified by RFLP and near-full-length 16S rDNA sequences. Approximately 48 operational taxonomic units (OTUs) were found among 139 positive clones from the superstratum sample; 26 among 149 were from the middle-level sample and 35 among 132 were from the substrate sample. Thermobifida fusca was common in the superstratum layer of the pile. Some Bacillus spp. were remarkable in the middle-level layer, and Clostridium sp. was dominant in the substrate layer. Among 109 OTUs, 99 displayed homology with those in the GenBank database. Ten OTUs were not closely related to any known species. The superstratum sample had the highest microbial diversity, and different and distinct bacterial communities were detected in the three different layers. This study demonstrated the spatial characteristics of the microbial community distribution in the cooling stage of swine manure compost.

• The Plant Pathology Journal
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• v.39 no.1
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• pp.149-157
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• 2023

Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrI and 16SrXXXII groups) in Jeju Island (Republic of Korea).

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1541-1545
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• 2006

The polymorphism of insulin-like growth factor I receptor (IGFIR) gene in 12 pig breeds (total n = 593) was detected by PCR-SacII-restriction fragment length polymorphism and allele A (379 bp) or allele B (235 bp and 144 bp) observed. In the studied breeds, it was found that European pigs principally carried allele A, while Chinese native pig breeds principally carried allele B. In addition, the role of pig IGFIR was investigated in 156 Wanbai pigs and 212 Large Yorkshire pigs. Growth related variables including body weight at birth, 2-, 4- and 6-mo of age and backfat thickness and lean percentage estimated by ultrasonography at 6-mo of age were recorded in analyzing the association between IGFIR gene polymorphism and growth traits. AA-genotype pigs exhibited greater (p<0.05) body weights (BW) at birth, 2- and 6-mo of age, but not at 4-mo of age, than those of the BB-genotype in Wanbai and Yorkshire breeds. Moreover, in the Yorkshire breed, AA-genotype pigs had less backfat thickness (p<0.05) and greater lean percentage (p<0.01) than the BB genotype. Based on these results, it is necessary to do more studies on IGFIR before introducing the IGFIR locus into breeding programs.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.171-176
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• 2005

Objective: To investigate whether polymorphism of CYP19 gene is associated with the risk of advanced endometriosis in Korean women. Methods: Blood samples were collected from 202 endometriosis patients and 221 controls. The patients with endometriosis of stages III and IV diagnosed by both pathologic and laparoscopic findings to according modified AFS classification. The women undergoing laparoscopic surgery or laparotomy for non-malignant lesions were included in the control group. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of PCR products were done to determine all individuals' genotype. Results: The heterozygous allele in CYP19 gene was the most common genotypes in both endometriosis and healthy control groups (52.0% vs. 46.1%). CYP19 gene polymorphisms did not show the significant differences between the control group and endometriosis group. Conclusion: The results suggested that the CYP19 genetic polymorphism was not associated with a risk of advanced endometriosis in Korean women.

• Korean Journal of Clinical Laboratory Science
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• v.45 no.2
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• pp.48-53
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• 2013

Recently, the isolation rate of nontuberculous mycobacteria (NTM) in clinical laboratories and the incidence of NTM infections are on the increase in Korea, but there have been only a few studies that reveal the general aspect of NTM isolation or species distribution. Therefore, this study was performed to examine the species identification by PCR-restriction fragment length polymorphism analysis (PRA, PCR-RFLP), and the clinical significance of mycobacterial cultures. PRA was used during the novel region of the rpoB gene and was developed for rapid and precise identification of mycobacteria to the species level. From January 2012 to April 2012, we examined pre-identified nontuberculous mycobacteria (60 species in 3 hospital of Busan-Kyeongnam area). We confirmed 4 (6.6%) Mycobacterium tuberculosis (MTB) and 56 (93.4%) NTM from 60 pre-identified NTM species by multiplex PCR (MolecuTech $MTB-ID^R$ V3, YD Diagnostics, Korea) and PRA (Myco-ID, YD Diagnostics, Korea). The distribution of 56 NTM species were M. intracellulare type I 15 (26.7%), M. avium 14 (25%), M. abscessus 11 (19.5%), M. kansasii type I 3 (5.4%), M. pulveris 2 (3.6%), M. intracellulare type, M. chelonae, M. kansasii type V, M. gallinarum, M. wolinskyi. Respectively, 1 (1.8%) and 6 (10.7%) species were not identified.

• BMB Reports
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• v.29 no.5
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• pp.442-447
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• 1996

Mutations in codon 12, 13 and 61 of one of the three ras genes, H-ras, K-ras and N-ras, convert these genes into active oncogenes. The presence of H-ras gene mutations have important prognostic implications in various cancers. In this study, the H-ras gene mutations were investigated by two-step PCRRFLP in patients with bladder and stomach cancer. For the control experiments, T24 and SK2 cell lines were used. In a total of 36 bladder cancer patient cases, five (13.9%) mutations were found by this method. Of these, point 12 mutations were two (5.6%) cases and point 61 mutations were three (8.3%) cases. On the other hand, H-ras mutation was not found in 29 cases of stomach cancer. The results of the mutated H-ras gene confirmed by direct sequencing analysis were correlated well with PCR analysis. From the sensitivity test, the H-ras mutation was found to have about 0.2% of mutated DNA mingled in normal DNA. In conclusion, the H-ras mutation has a higher clinical Significance in bladder cancer than stomach cancer. Moreover the two-step PCR-RFLP method is sensitive, rapid and relatively simple for clinical work in detecting H-ras point mutations.

• Parasites, Hosts and Diseases
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• v.47 no.4
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• pp.413-415
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• 2009

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5' and 3' ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.

• Journal of Microbiology
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• v.44 no.1
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.778-785
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• 1999

To detect CDV RNA in clinical samples and differentiate prevailing CDV virulent strains affecting susceptible animals from attenuated vaccine strains, we performed RT-PCR, RFLP, and sequencing. CDV specific primers were generated from the middle part of nucleocapsid gene. The expected size of PCR products, 519 bp, was observed in tissues of Jindo dog, poodle dog, badger, fourteen of nineteen blood samples as well as 5 vaccine strains including domestic and imported products. The PCR products obtained from tissues and PBMCs of infected animals were digested to 317- and 202-bp fragments by Bam HI, but the products obtained from four of five vaccine strains and Lederle strain were not digesed by Bam HI. Only one vaccine strain of which the PCR products were digested by Bam HI was confirmed as imported vaccine, modified Synider Hill strain. Based on seqencing data obtained from the 519-bp products, it was confirmed that Bam HI restriction site tends to be conserved in field isolates compared to the commercially available attenuated vaccine strains. Partial nucleotide sequences of CDV NP gene obtained from tissues of Jindo dog, poodle and badger shared 100% homology each other, whereas the nucleotide sequences showed 96.3, 96.5, 93.6 and 93.4% homology with Yanaka (virulent), Han95 (virulent), Lederle (attenuated) and Onderstepoort (attenuated) strain, respectively.