• 식물병연구
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• 제23권1호
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• pp.65-68
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• 2017

2단계의 nested PCR 방법을 이용하여 보리 및 벼 재배 토양에서 Barley yellow mosaic virus (BaYMV)를 검출하였다. BaYMV 분절 RNA1 외피단백질 영역의 특이 프라이머로 1차 PCR을 하고 내부서열로부터 작성된 프라이머로 2차 PCR을 실시하여 확보된 372 bp의 PCR 산물이 BaYMV 외피단백질 영역과 98%-100% 염기서열이 일치하여 BaYMV를 검출할 수 있음을 확인하였다. 이 결과는 토양으로부터 BaYMV 검출에 관한 최초의 보고이며 토양전염성 바이러스의 정확한 진단과 예찰에 적용될 수 있을 것으로 생각한다.

• 대한소아치과학회지
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• 제25권2호
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• pp.292-303
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• 1998

The arbitrary primer polymerase chain reaction(AP-PCR) and Southern blot restriction fragment length polymorphism(RFLP) were used to genotype the cariogenic pathogen S. mutans in children. Following the morphologic chracteristics of colony on selective medium for S. mutans, total genomic DNA from 155 strains was extracted by conventional methods. Among 155 strains, 143 strains (92.3%) were confirmed S. mutans by PCR with dexA gene and 114 strains were used in this study. Three random sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 2% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI and BamHI, separated on a 0.7 % agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterised 1.6 kilobases (kb) DNA fragment cloned from gtf B gene of S. mutans. The probe was labeled with isotope[$^{32}P-{\alpha}CTP$], and hybridized fragments were detected with intensifying screen. AP-PCR produced 4-8 DNA bands in the 0.25-10 kb regions and distinguished 9, 10 or 12 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 2 hybridization patterns consisting of 1 DNA fragments 450, 500 bp. These results indicate that AP-PCR is more discriminative method for genotyping of S. mutans.

• Animal cells and systems
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• 제1권1호
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• pp.129-134
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• 1997

HLA-A2 is one of the most diversified HLA-class I antigen with 17 subtypes so far identified at the molecular level. HLA-A*02 subtyping has significant implications on the tissue typing for organ and bone marrow transplantations. Recently, DNA-based typing methods have been successfully applied to the elucidation of HLA gene polymorphisms. In the present study, HLA-A*O2 genotyping was established by using nested polymerase chain reaction-sequence specific primers (PCR-SSP) and distribution of A*O2 alleles were determined in Korean individuals. Genomic DNA prepared from four B-lymphoblastoid cell lines and lymphocytes from serologically defined 48 HLA-A2 Korean individuals by phenol/chloroform extractions was typed. The results of the four B-lymphoblastoid cells were consistent with the previous data typed by PCR analysis. Five A*O2 alleles-A*0201, A*0203, A*0206, A*0207 and A*0210-were commonly observed in a total of 17 A*02 alleles. Of these, A*0207 (f=49.0%) was the most frequent allele in Korean population. A*0206 (f=28.3%) and A*0201 (f=17.0%) were also found frequently while A*0203 and A*0210 types were observed in less than 5%. In conclusion, the high level of discrimination for HLA-A*O2 alleles will prove useful and informative in the study of transplant survival, and may identify the importance of allelic differences not readily detectable by serology on host and donor compatibility.

• 한국자원식물학회지
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• 제28권3호
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• pp.341-349
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• 2015

Magnoliae Flos (Sini in Korean) is one of the most important oriental medicinal plants. In the Korean Herbal Pharmacopeia, the bud of the all species in Manolia denudate and Manolia genus were regarded as the botanical sources for ‘Sini’. Most the dried bud of Manolia denudata, Manolia biondii and Manolia sprengeri were used as ‘Xin-yi’ in China. Therefore, the purpose of this study was to determine and compare the ‘Magnolia’ species, four species including Manolia denudata, M. biondii, M. liliiflora and M. Kobus were analysis of sequencing data revealed DNA polymorphisms. The based on tRNA coding leucine/phenylalanine (trnL-F) and NADH-plastoquinone oxidoreductase subunit 5 (ndhF) sequences in chloroplast DNA. For the identification of ‘Magnolia’ species, polymerase chain reaction (PCR) analysis of chloroplast DNA regions such as ndhF have proven an appropriate method. A single nucleotide polymorphism (SNP) has been identified between genuine “Sini” and their fraudulent and misuse. Specific PCR primers were designed from this polymorphic site within the sequence data, and were used to detect true plants via multiplex PCR.

• Parasites, Hosts and Diseases
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• 제44권2호
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• pp.101-116
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• 2006

Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vagina/is. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and $GRO-\alpha$, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.

• Food Science and Biotechnology
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• 제17권4호
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• pp.888-891
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• 2008

Twenty-three fungal strains were isolated from meju that had originated from the Sunchang province, the famous location for making fermented soybean foods in Korea. The restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rDNA (ITS-RFLP) was applied to differentiate the isolated fungal strains. First, the ITS region by polymerase chain reaction (PCR) with specific primers was amplified and then cleaved the products with different restriction enzymes. Cleavage of the amplified fragments with the restriction enzymes AluI, HaeIII, HhaI, and TaqI revealed extensive polymorphisms. The ITS-RFLP results highly correlated with ITS sequence analysis. All of the 23 fungal strains were classified into 5 groups by ITS-RFLP analysis. Aspergillus oryzae was the major fungal strain isolated from Sunchang meju (12 out of 23), while Aspergillus fumigatus was the next most frequently isolated strain (7 out of 23). In contrast, it was found that Fusarium asiaticum, Aspergillus sydowii, and Arthrinium sp. were the minor fungal strains in meju.

• 농업과학연구
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• 제45권4호
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• pp.575-582
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• 2018

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is a devastating disease found in many cucurbits cultivation fields. The genetic diversity for 29 strains of A. citrulli collected from various cucurbits in South Korea was determined by DNA fingerprinting with a pathogenicity test, multi locus analysis, Rep-PCR (repetitive sequence polymerase chain reaction), and URP (universal rice primers) PCR bands. Two distinct groups (Korean Clonal Complex, KCC1 and KCC2) in the population were identified based on group specific genetic variation in the multi locus phylogeny using six conserved loci and showed a very high similarity with DNA sequences for representative foreign groups [the group I (CC1-1 type) and the group II (CC2-5 type)] widely distributed worldwide, respectively. Additionally, in the case of phaC, a new genotype was found within each Korean group. The KCC1 was more heterogeneous compared to the KCC2. The KCC1 recovered mainly from melons and watermelons (ratio of 6 : 3) and 15 of the 20 KCC2 strains recovered from watermelons were dominant in the pathogen population. Accordingly, this study found that two distinct groups of differentiated A. citrulli exist in South Korea, genetically very similar to representative foreign groups, with a new genotype in each group resulting in their genetic diversity.

• 한국산림과학회지
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• 제108권3호
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• pp.302-310
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• 2019

정확하고 신속한 품종식별은 효율적인 육종과 육종가의 권리 보호를 위하여 필수적이다. 대추나무 품종을 구분하는 전통적인 방법은 형태적 특성을 근거로 하지만, 시기적 제약과 환경의 영향으로 형태적 형질만을 이용하여 구별하기에는 어려움이 있다. 이에 본 연구에서는 ISSR 분석으로 얻어진 단편을 복제하여 안정적인 식별을 위한 SCAR 표지를 개발하였다. 대리조와 보은대추 품종에서 특이성을 보이는 ISSR 밴드를 대상으로 정제, 복제, 염기서열 분석을 수행함으로써 827Dalizao550, 827Boeun750, 846Boeun700, 847Dalizao850로 명명된 4개의 클론을 확인하였다. 대추나무 품종 특이성 분석을 위한 4쌍의 SCAR 프라이머를 제작하였으며, 대추나무와 묏대추나무를 포함하는 32개체에 대하여 PCR 분석을 수행하였다. 827Dalizao550 SCAR 표지의 PCR 결과 6개체(대리조, 산동이조, 동조, 원령 신 2호, 묏대추나무 2, 묏대추나무 4)에서 550 bp의 특이적인 밴드가 증폭되었고, 나머지 개체에서는 예기치 않은 밴드(490 bp)가 증폭되었다. 또한 847Dalizao850 SCAR 표지의 PCR 결과 대리조, 산동이조, 동조에서 850 bp의 밴드가 나타났고 예기치 않은 900 bp의 밴드가 5개체(파조, 묏대추나무 1, 묏대추나무 2, 묏대추나무 3, 묏대추나무 4)에서 증폭되었다. 이상의 결과로 보아 새롭게 개발된 표지들은 대추나무 품종식별을 위한 빠르고 신뢰성 있는 수단으로 이용될 수 있을 것으로 생각된다. 하지만 보다 다양한 품종들의 식별을 위해서는 다형성 정보의 추가와 SCAR 표지의 개발이 필요하다.

• 대한의생명과학회지
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• 제17권3호
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• Journal of Animal Science and Technology
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• 제45권6호
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• pp.911-916
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• 2003

돼지 Duroc 품종의 mitochondria DNA D-loop전체 유전자를 증폭하기 위하여 많은 동물에서 고도로 상동성이 높은 tRNA-Pro와 tRNA-Phe 염기서열 일부를 이용하여 oligonucleotide primer를 제작하였다. 그 결과 Duroc 품종의 D-loop 전체 유전자는 1,145 base pairs 였으며, 그 중간위치에 10bp의 Sus Scrofa-specific sequence (TACACGTGCG)가 10개 존재하고 있었다. 돌연변이 검출을 위하여 가장 변이가 심한 지역을 primer 제작하여 345 bp의 DNA 단편을 증폭하였으며, Single Stranded Conformation Polymorphism(SSCP) 분석은 8% polyacrylamide gel에서 200 V, 16시간 전기영동하여 ethidium bromide (EtBr)로 10분간 염색하여 UV image analyzer로 관찰하였다. 그 결과 두 개의 서로 다른 밴드유형을 관찰하였으며, 21개 부위에서 염기서열 변이가 관찰되었다. 이러한 결과는 유전적 다양성 변이를 검출하는데 SSCP 분석이 유용한 도구라고 사료된다.