• Biomedical Science Letters
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• v.15 no.4
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• pp.295-300
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• 2009

Tuberculosis caused by Mycobacterium tuberculosis (MTB) still remains to be the most dreadful infectious disease affecting almost every country. In the present study, we developed a simple and rapid but accurate and sensitive assay method for detecting MTB using microplate hybridization assay. For this, a selective region of the rpoB gene was used to design PCR primers, and MTB and Mycobacterium genus-specific probe molecules. The specificity of the assay was confirmed using fifteen different mycobacterial reference strains and twelve different non-mycobacterial reference strains, and the sensitivity was determined to be 100 fg using genomic DNA of MTB reference strain, H37Rv. Subsequently, a total of 62 sputum samples with diverse smear scores and culture positive results were used to evaluate the kit performance. In brief, the specificity and the sensitivity of the assay were 100% and 98.4%, respectively.

• The Transactions of the Korean Institute of Electrical Engineers C
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• v.51 no.6
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• pp.265-270
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• 2002

The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and application area. So, the improvement of DNA hybridization detection method is very important for the determination of this hybridization reaction. Several molecular biological techniques require accurate predictions of matched versus mismatched hybridization thermodynamics, such as PCR, sequencing by hybridization, gene diagnostics and antisense oligonucleotide probes. In addition, recent developments of oligonucleotide chip arrays as means for biochemical assays and DNA sequencing requires accurate knowledge of hybridization thermodynamics and population ratios at matched and mismatched target sites. In this study, we report the characteristics of the probe and matched, mismatched target oligonucleotide hybridization reaction using thermodynamic method. Thermodynamic of 5 oligonucleotides with central and terminal mismatch sequences were obtained by measured UV-absorbance as a function of temperature. The data show that the nearest-neighbor base-pair model is adequate for predicting thermodynamics of oligonucleotides with average deviations for $\Delta$H$^{0}$ , $\Delta$S$^{0}$ , $\Delta$G$_{37}$ $^{0}$ and T$_{m}$, respectively.>$^{0}$ and T$_{m}$, respectively.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.48-48
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• 2003

본 실험은 spermatogonia 단계에 발현하는 유전자를 찾기 위하여 suppression subtractive hybridization를 수행하였다. 기존에 mouse에서는 spermatogonia 특이적인 유전자들이 밝혀져 있기 때문에 pig에 특이적인 유전자를 찾기 위하여 pig 250days testis와 pig 60days testis를 재료로 하여 실험하였다. SSH를 통하여 254days testis에 특이적으로 발현되는 후보유전자를 7개 찾았고 25days testis와 60days testis 의 Northern blot을 통하여 25days에 과발현하고 60days에 발현의 양이 대폭 줄어드는 spermatogonia 유전자로 생각되는 후보유전자 2개를 선택하여 pig tissue northern blot, genomic DNA southern blot, RT-PCR 그리고 In-situ hybridization을 수행하였다. Tissue northern blot과 RT-PCR을 통하여 후보자 1번은 간과 폐, 난소, 정소에서 발현하고, 후보유전자 15번은 난소와 정소에서만 특이적으로 발현함을 알았다. DNA sequence analysis와 NCBI Blast search를 통하여 후보자 1번은 다른 종에서 밝혀진 유전자였고 후보유전자 15번은 어느 종에서도 밝혀지지 않은 새로운 유전자였다. Degenerated primer를 통하여 후보자 1번의 pig full sequence를 밝히고 NCBI에 등록하였다. 그리고 In-situ hybridization을 통하여 후보유전자득이 20일째 testis의 Leydic cell에서 많이 발현되고 adult testis에서는 발현이 감소하는 결과를 얻었다. 이것으로 보아 위의 두 후보유전자는 spermatogonia에 직접 관련된 유전자이기 보다는 spermatogonia의 발달에 영향을 주는 leydic cell 특이발현을 가진 유전자로 사료되어진다.

• Plant Breeding and Biotechnology
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• v.5 no.4
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• pp.269-281
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• 2017

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.3
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• pp.120-123
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• 2011

A total of 30 water samples were collected from 30 different public baths in Seoul, Korea. Contamination of public bath water by waterborne pathogens can cause disease outbreaks and contribute to increase background rates of disease. Pathogens in water was filtered by nitrocellulose membrane with $0.45{\mu}m$ pore size. The membrane filters were analyzed by both cultivation and polymerase chain reaction (PCR) amplification of partial 16S rRNA gene. Various microorganisms including 4 Escherichia coli/Shigella spp. 1 Salmonella spp. 3 Pseudomonas aeruginosa and 2 Mycobacterium spp. were identified by reverse blot hybridization assay (REBA). PCR-REBA was able to identify many bacterial genera in one assay. Our results suggest that appropriate hygiene practice and continuous monitoring is needed for reducing health risk associated with public bath houses.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.589-593
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• 1998

DNA amplification by polymerase chain reaction (PCR) was used to specifically detect Plasmodiophora brassicae, causing clubroot of crucifers. On the basis of DNA sequence informations, an oligonucleotide primer set specific for the pathogen was designed form small subunit gene (18S-like) and internal transcribed spacer (ITS) region of ribosomal DNA. Primer ITS 5/PB-C produced an amplification product of approximately 520 bp in length with DNA from P. brassicae. However, no amplification product was produced with DNAs from several soil-borne fungi, Didymella bryoniae and Rhizopus stolonifer. Using these primers, the clubroot pathogen was readily detected from infected roots of crucifers, but not from healthy roots. Southern hybridization analysis further confirmed that the amplification product was originated from P. brassicae.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.175-182
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• 1998

We investigated the rate of hepatitis G virus infection among 50 patients who were not infected with the hepatitis C virus but showed symptoms of hepatitis. Viral RNA was extracted from the patients' sera and cDNA was synthesized and amplified by RT-PCR (reverse transcription-polymerase chain reaction) using random hexamer and 5 primers (470-20-1-77F, 470-20-1-211R, 470-20-1-211R-biotin, GV57-4512MF, GV57-4657MR). The amplified PCR products were confirmed by electrochemiluminescence (ECL), liquid hybridization (LH) and Southern blotting (SB). Among the 50 PCR products, by means of ECL, we found 4 samples to be positive and 5 samples to be indeterminate. The GV45-89M probe (5'-CYCGCTGRTITGGGGTGTACfGGAAGGC-3') was end-labelled with gamma-$^{32}P$ ATP and used for liquid hybridization with the PCR products. By using liquid hybridization, we detected specific bands from 4 positive sera and also from one indeterminate serum as determined by ECL. An 1.5% agarose gel electrophoresis of the 9 PCR products which were HGV positive or indeterminate as determined by ECL showed a 160bp band from 4 positive and one indeterminate serum. The 5 PCR products proved to be positive when SB was applied with the GV45-89M probe as well as when LH was applied. LH and SB were shown to have higher sensitivity and specificity than ECL. Two cases among 5 positive cases had relatively high SGOT, SGPT, ALP values when compared with other 48 cases. In summary, we confirmed hepatitis G virus infection in 5 cases among 50 Korean patients showing symptoms of viral hepatitis.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.515-530
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• 1995

Coronaviridae에 속하는 transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)를 specific하게 detection할 수 있는 방법을 개발하고자 본 연구를 수행하였다. 두 바이러스 모두 RNA 바이러스이기 때문에 reverse transcription-polymerase chain reaction(RT-PCR)으로 nucleocapsid(N) protein gene의 cDNA를 증폭시켰다. SmaI으로 처리한 pTZ19R에 ligation시킨 후 염기서열을 밝히고자 sequencing하였다. 각각의 prototype virus와 비교하여 상동성을 밝혔다. 두 바이러스에 대한 cDNA probe를 제작하여 Southern blot hybridization을 실시하였다. TGEV의 경우 백신주인 P45와 병독주인 Miller strain을 사용하였다. cDNA를 증폭시키기 위해 N1/N1R과 N2/N2R 두 가지 primer를 이용한 결과, N1/N1R primer의 경우 586bp 크기의 PCR product를 얻을 수 있었고, N2/N2R primers로 582bp의 cDNA를 증폭시킬 수 있었다. PEDV 실험을 위하여 PED 임상 증상을 나타내는 분변을 이용하여 RT-PCR을 실시하였다. P2/P2R primer로 753bp의 PCR product를 얻을 수 있었다. TGEV의 두 가지 strain의 N protein gene을 sequencing하여 prototype인 Purdue strain과 염기서열 상동성을 조사한 결과, 97%이상의 높은 homology를 나타내었다. PED-V 역시 N protein gene을 sequencing하여 CV777과 염기서열 상동성을 조사한 결과 97%이상의 homology로 PEDV임을 알 수 있었다. TGEV와 PEDV의 염기서열을 비교한 결과 29%의 낮은 homology를 관찰할 수 있었다. 두 가지 바이러스의 N protein gene에 대한 cDNA probe를 제작하여 Southern blot hybridization을 한 결과, 각 바이러스에 매우 특이적 반응을 나타내었다.

• Korean Journal of Veterinary Service
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• v.40 no.3
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• pp.161-168
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• 2017

Caprine arthritis encephalitis (CAE) virus is a causative agent of caprine arthritis-encephalitis. In our previous study we reported a prevalence of CAE. In this study, we described the further detailed pathological features of CAE and examined the detection of virus by in situ hybridization (ISH). Histopathologically, interstitial pneumonia and bronchopneumonia in lung, focal inflammation in mammary glands, perivascular cuffing in brain, arthritis, and focal necrosis, mild steatosis, inflammatory cell infiltration of liver were noted. CAEV proviral-DNA was identified by nested polymerase chain reaction (PCR) in blood cells, brain, synovial fluid, and lymph node. Confirmation by nested PCR involved amplification of a 296 bp ($1^{st}$ PCR) and 185 bp ($2^{nd}$ PCR) fragments corresponding to a conserved region on the gag gene of CAEV. Positive ISH signals were detected in the brain and liver. In conclusion, significant histopathological findings included parenchymal infection in various organs, including the lung, liver, brain, joint, and mammary gland were noted in the CAEV infected dairy goat. ISH can help confirm the diagnosis of CAE in formalin-fixed samples.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.111-118
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• 1997

Four separate pairs of oligonucleotide primers within the coding region in a T sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme 3nd Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. morginnle and 3. ouata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive. PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.