• Journal of environmental and Sanitary engineering
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• v.14 no.4
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• pp.99-109
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• 1999

Enteropathogenic Yersina enterocolitica is an important cause of human and animal disease. Phenotypic and genotypic characteristics currently used to identify Yersinia enterocolitica are not necessarily sufficient to differentiate pathogenic from non-pathogenic strains or to analyze the epidemiology of yersiniae at a molecular level. To improve the characterization of Yersinia enterocolitica, A total of 65 isolates of Yersinia enterocolitica were examined with bioserotyping, antibiotic susceptibilities, PFGE, PCR-ribotyping. Genomic DNA pattern generated by PFGE are highly specific for different strains of an organism and have significant value in epidemiologic investigations. The PFGE analysis of Not I-digested chromosomal DNA of Y. enterocolitica were performed with a CHEF Mapper(Bio-Rad, USA). Not I generated 19 restriction endonuclease digestion profiles(REDP). PCR-ribotyping, performed with primers complementry to conserved regions of 16S and 23S rRNA gene, generated 13 ribotypes. PCR-ribotyping can be considered a good technich for subtyping strains of Y.enterocolitica.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.24-37
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• 2005

This study was conducted to analyze the molecular epidemiological properties and to select the most efficient and reliable PCR method on 116 of Staphylococcus aureus (S. aureus) isolates from Korean cattle, black goat, pig, dog, chicken, mouse and also human clinical cases from hospital. The distribution patterns of SSG [species specific genes; coagulase (coa), protein A (spa), nuclease (nuc) and aroA (RsaI) gene] were analyzed by PCR method. Among the SSGs, the nuc-gene was found in all strains $(100\%)$ tested and followed by coa-gene $(87.9\%)$, spa-gene $(91.4\%)$ and aroA-gene $(26.7\%)$, in order. The genetic subtyping by RFLP method was performed on the coa [AluI] and aroA-gene [RsaI] PCR products. The mecA-gene PCR and PCR-RFLP techniques were chosen to detect and verify of MRSA strains. Only the human strains $(12.1\%)$ were detected the positive mecA-gene products (533 bp), which were divided into two specific bands [201 & 332 bp] by HhaI enzyme digestion. On coa-gene and spa-gene typing, coa-gene was typed with ten kinds of genotype and coa-3 type were determined as the most predominant genotype, while spa-gene was divided into eleven kinds of genotype and also spa-7 type were selected the most prevalent genotype based on their genetic variations. On the aroA and coa-gene subtyping by PCR-RFLP, aroA-gene products were discriminated with only seven types of genotype, while coa-gene products were further divided into an eleven genotype, respectively. In comparison of SID values of five PCR based typing methods, the coa-PCR-RFLP (SID0.894) was evaluated the most efficient and reliable tools and followed by coa-PCR (SID0.883) and aroA-PCR-RFLP (SID0.462), in order. In conclusion, we could determined that the coa-PCR-RFLP method was the most suitable genetic analysis tool for S. aureus and MRSA strains from domestic animals and humans.

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.533-537
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• 2004

In this study, we performed a PCR-RFLP analysis of NADH oxidase gene(nox) for the characterization of porcine intestinal spirochetes isolated from Korea by the comparison with Brachyspira hyodysenteriae and B. pilosicoli reference strains. Eleven strains including four reference strains, B. hyodysenteriae B204, B234, B169, B. pilosicoli P43/6/78 and seven Korean isolates were used. PCR products of 939 bp were amplified using nox-specific primers and digested with two restriction enzymes, Bfm I and Dpn II. In study using Bfm I, both strains showed no difference in fragmented size(197 and 741 bp). When use Dpn II, B. hyodysenteriae showed two bands(209 and 684 bp), however B. pilosicoli showed a single band of 896 bp. Our results indicate that nox-specific PCR-RFLP could be used as a typing method of Brachyspira species and as an epidemiological method for identifying spirochetes isolated from swine.

• Journal of Food Hygiene and Safety
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• v.29 no.3
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• pp.223-227
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• 2014

This study was investigated to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) isolated from raw milk samples and to further study on the molecular characteristics of the MRSA isolates. Using Staphylococcus Medium 110, Staphylococcus spp. were isolated from raw milk samples and further identification was carried by Vitek2 system. Minimum inhibitory concentrations (MICs) of antibiotics were conducted by serial dilution method according to the Clinical Laboratory Standards Institute (CLSI) guideline. For the detection of resistance genes and molecular characterization, PCR reaction was performed by gene specific primers and followed by DNA sequencing. Of the 698 milk samples, 94 Staphylococcus aureus (S. aureus) were identified (94 S. aureus/286 Staphylococcus spp.). Of the 94 S. aureus, seven isolates have mecA, a methicillin resistant gene. mecA positive seven isolates were then characterized by staphylococcal cassette chromosome mec (SCCmec) typing, and Panton-Valentine Leukocidin (pvl) gene using PCR. All of mecA positive isolates were resistant to ampicillin and oxacillin, but sensitive to teicoplanin, vancomycin and ciprofloxacin. One of seven isolates was SCCmec type II and six isolates were type IV and all seven isolates were pvl gene negative.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1336-1344
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• 2011

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

• The Korean Journal of Mycology
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• v.49 no.4
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• pp.455-467
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• 2021

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH₄)₂SO₄ after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.165-174
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• 1998

Characterizations of the VP4 (P type) and VP7 (G type) genes of Korean isolates of bovine rotavirus were performed using RT-PCR/RFLP and nucleotide sequencing analysis. After RT-PCR amplification of partial length (1094bp) of the VP4 and full length (1062bp) of the VP7 genes, amplified PCR products were digested with restriction endonucleases and digestion patterns were compared with those of reference rotaviruses. With the VP4 genes, four RFLP (A-D) profiles were observed; three (A, Band C) were the same as those of bovine rotavirus NCDV (P[1]), IND (P[5]) and B223 (P[11]), respectively. Profile D was the same as that of porcine rotavirus OSU (P[7]). With the VP7 genes, five RFLP profiles (I-V) were observed; three of them (I, II and III) were the same as those of bovine rotavirus NCDV (G6), Cody 1-801 (G8), and B223 (G10), respectively. Profile IV and V were atypical to those of reference bovine rotaviruses used in this study. These two profiles were identified as G6 and G5, respectively, after analyzing and comparing the nucleotide sequences. The G typing analysis revealed that 61.9% (26/42) were G6, which included G6 subtype; 28.6% (12/42) were G5; 7.1% (3/42) were G10; 2.4% (1/42) were G8. The P typing analysis revealed that 54.8% (23/42) were P[5]; 28.6% (12/42) were P[7]; 11.8% (5/42) were [11]; 4.8% (2/42) were P[1]. Our results showed that G6/P[5] were the most prevalent rotaviruses in diarrheic calves in Korea. Also, this is the first report that G5/P[7] rotaviruses were identified from cattle with diarrhea.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.221-227
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• 2000

This study was performed to investigate the biological characteristics of twenty isolates of extended spectnlm $\beta$-lactamase (ESBL) producing Klebsiellapnezm~onia collected kom the various clinical specimens of three hospitals in Pusan. Isoelectric focusing (IEF) and PCR were used to determine the types of $\beta$-lactamase gene in this study. Twenty isolates of ESBL producing Klebsiellnp~ieun~or~iae could be divided by PCR, such as TEM type (I1 strains), SHV type (8 strains); non TEM non SHV type (1 strain). In the isoelechic focusing test, the PI of TEM type was 5.2-6.0 and that of SHV type was 6.9-7.4. According to the pI value and PCR bands, twenty strains of ESBL Klebsiellapneumoniae were divided into 5 types: TEM type @I 5.2-6.0; 1080 bp on PCR band), TEM + SHV type (pI 5.2-6.0; andpI 7.0-7.4; 1080 bp and 599 bp on PCR band), SHV type (pl7.0-7.4; 599 bp on PCR band), non TEM non SHV type, and otber type (PCR result was SHV type but pI was not detected).

• Clinical and Experimental Pediatrics
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• v.46 no.5
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• pp.467-473
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• 2003

Purpose : The Epstein-Barr virus(EBV), gamma herpesvirus, is an important pathogen that is widespread around the world. The EBV causes various diseases depending on the geographic location, and on the immunity or the premorbid condition of the person exposed to EBV. To evaluate EBV typing may be the most important step to figure out the pathogenesis of EBV associated diseases, and we need to re-evaluate the pathologic role of human leukocyte antigen(HLA) in developing Epstein- Barr virus associated acute infectious mononucleosis by using newly developed methods. Methods : This study included 24 children(age range : 6 to 13 years), serologically confirmed with acute infectious mononucleosis. The control group for the HLA type consisted of 200 age-matched healthy children. To classify HLA I, modified ARMs-PCR was used, while modified PCR-SSOP was utilized in typing of HLA II. Also, we performed EBV typing in study patients by using a one-step PCR. Results : The results of HLA types : In HLA class I, HLA-A24 was positive in 69 of 200 healthy children and positive in 14 of 24 patients in the study group(relative risk : 3.5724, chi-square; 5.26, P<0.05). In HLA class II, HLA-DRB1*07 was detected in 18 of 200 healthy children, and eight of 24 patients in the study group(relative risk; 506173, chi-square; 9.73, P<0.01). The results of EBV types : In the research group, 20(83.8%) of 24 patients were shedding type A virus, while 4(16.7%) were type B. Conclusion : We conclude that development of infectious mononucleosis may be associated with HLA types, and these results suggest that acute infectious mononucleosis could have hereditary traits. And we confirm that type A EBV is highly prevalent in patients with acute infectious mononucleosis in Korea. Also, our results suggest that further large scale studies, including adult groups, regarding the association between pathogenesis of EBV with HLA-DP or HLA-DQ will be warranted.

• Proceedings of the Zoological Society Korea Conference
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• 1998.10b
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• pp.346.2-346
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• 1998

No Abstract, See Full Text