• 대한임상검사과학회지
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• 제50권3호
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• pp.261-266
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• 2018

만성C형 간염환자 160예를 대상으로 immunoblot방법과 RT-PCR-hybridization법을 실시하여 임상검사의 유용성을 알아보았다. 양성 150예 중 133예만 RT-PCR-hybridization 법에서 양성을 나타내어 immunoblot법의 진양성율은 88.6%를 나타났으며 두 방법 간의 일치율은 89.3%를 나타났다. 한국인의 HCV 아형을 알기 위하여 혈청형과 유전형을 실시하였다. HCV혈청형 1형과 2형 그리고 1b와 2a유전형이 가장 많은 C형간염 감염원으로 나타났다. 49예를 혈청형과 유전형을 서로 비교한 결과, 혈청형 검사에서 28예(57.1%)가 1형, 21예(42.9%)가 2형으로 나타났으며 유전자형 검사에서 1b형이 25예(51.0%), 1b/2b를 나타낸 예가 1예(2.0%), 2a형이 17예(34.7%), 2a/2c를 나타낸 예가 4예(8.2%), 그리고 2b형이 2예(4.1%)로 나타났다. 본 연구에서는 HCV 간이검출법으로는 immunoblot방법이 유용하며, immunoblot방법 양성 확진검사로는 RT-PCR-hybridization법을 실시하였다. 혈청형이 C형간염의 치료 나 진행과정을 관찰하기 위해서는 유전형보다 유용하나 인터페론 치료효과는 1형과 2형 혈청형별에 따라 차이를 보이지 않았다.

• 대한수의학회지
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• 제42권3호
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• pp.351-362
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• 2002

Newcastle disease (ND) is a highly contagious infection of poultry, Two pathology-based techniques, in situ RT-PCR and in situ hybridization (ISH) were applied to formalin-fixed, paraffin-embedded tissues from chickens naturally infected with velogenic ND virus (VNDV). Two pairs of primers and a probe for ISH and in situ RT-PCR, respectively, were selected from highly conserved region of matrix gene of NDV. The ISH experiment was carried out using MicroProbe$^{TM}$ capillary action system within 2 hours. In situ RT-PCR was performed using MicroProbe$^{TM}$ capillary action system and GeneAmp In Situ PCR system. With ISH and in situ RT-PCR, viral nucleic acid was detected in the central nervous system of chickens from infected with neurotropic velogenic Newcastle disease virus (NVNDV), whereas viral nucleic acid was detected in various organs or tissues of chickens from infected with viscerotropic velogenic Newcastle disease virus (VVNDV). In the NVND group, positive signals were characteristically defined in the cytoplasm of neuron, vascular endothelial cells, and perivascular mononuclear macrophages in the central nervous system. One of NVND group, chicken from one farm exhibited positive signals in the bronchial epithelium. The VVND group chickens showed positive reaction in the macrophages, vascular endothelium, and bronchiolar epithelium. Markedly, viral nucleic acid was detected in the macrophages of morphologically normal tissues which were peripheral or located in distant areas from lesions. The central nervous system of chickens infected with VVND virus had positive signals in the vascular endothelial cell, perivascular mononuclear macrophages and some neuron. The number and intensity of the positive cells by in situ RT-PCR were more and stronger, respectively, in comparison with those by ISH. Particularly, positive reaction was detected in macrophages infiltrating in cardiac muscle by in situ RT-PCR, but not obtained by ISH. Therefore, these results demonstrated that ISH is a rapid diagnostic method for detection of NDV and in situ RT-PCR can be used as an efficient method for detection of low viral load infection or subclinical viral infection of NDV.

• 식물조직배양학회지
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• 제27권3호
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• pp.169-173
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• 2000

Amaranthus hypochondriacus의 저장 단백질을 encoding 하는 AmA1 유전자를 RT-PCR 방법을 이용하여 분리하고 특성화하였다. AmAl 유전자를 담배에 형질전환 시키기 위해 CaMV 35S promoter와 3'NOS를 가지고 있는 식물 발현 vector에 subcloning 하고 이 재조합 vector를 이용하여 Agrobacterium-mediated형질전환 방법을 이용하여 담배에 도입시켰다 신초는 0.1 mg/L NAA, 1.0 mg/L BA, 100 mg/L kanamycin 그리고 250 mg/L cefotaxime이 첨가된 MS 선발 배지에서 선발했고, 선발된 신초는 식물 생장 조절제를 첨가 하지 않고 200 mg/L kanamycin과 250 mg/L cefotaxime이 첨가된 MS배지에서 뿌리를 유도하였다. 선발된 담배의 게놈내의 AmA1 유전자의 존재는 PCR 방법과 hybridization을 이용하여 확인되었고, AmA1 유전자의 발현은 RT-PCR방법과 Southern blot hybridization을 사용하여 확인되었다.

• BMB Reports
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• 제40권6호
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• pp.986-1001
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• 2007

Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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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.

• 대한의생명과학회지
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• 제21권4호
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• pp.227-232
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• 2015

Epstein-Barr virus (EBV) has a pathogenic role in several lymphomas including diffuse large B-cell lymphoma (DLBCL). In this study, we detected EBV from formalin-fixed paraffin embedded (FFPE) tissues of DLBCL patients by RT-PCR and compared the sensitivity of the RT-PCR method to in situ hybridization (ISH) method. The RNA was extracted from 91 FFPE samples with DLBCL and amplified with primers targeting EBV-encoded small RNA (EBER) by RT-PCR. When using the RT-PCR method, 13 of 91 patients (14.3%) were positive and among these 13 cases, 7 cases (7.7%) were from > 50-year-old patients that is classified as EBV positive DLBCL of the elderly. In previous results using ISH method, 3 of 91 patients (3.3%) were positive and 2 case (2.4%) were older than 50-year-old. These results indicate that RT-PCR method used in this study shows a higher sensitivity than ISH method. The ratio of male versus female among the EBV positive samples was 1.2:1 with the ratio of male higher. If RT-PCR method having high sensitivity is used simultaneously as well as the ISH method providing the information of the EBV positive cellular location, it is expected that EBV will be more accurately detected.

• 한국어병학회지
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• 제19권3호
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• pp.189-195
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• 2006

A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.

• 한국수의병리학회:학술대회논문집
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• 한국수의병리학회 2003년도 추계학술대회초록집
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• pp.26-26
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• 2003

Hepatitis E virus (HEV) has been recognized as a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries. The taxonomy of HEV is not clear and the virus remains unclassified. The objective of this study was to optimize conditions and procedures to detect swine HEV in formalin-fixed, paraffin-embedded tissues by nested RT-PCR and compare this detection method with in situ hybridization. (omitted)

• 대한수의학회지
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• 제35권3호
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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.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.285-292
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• 2009

Zoysiagrass (Zoysia japonica) is one of the important turfgrass species. Extending green period of zoysiagrass via delaying leaf senescence will make this species have more potential in the turfgrass industry. In this study, we found that zoysiagrass seedlings treated with $GA_3$ could delay the leaf senescence induced by darkness. To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-$non-GA_3-treated$ and $GA_3-treated$ seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Differentially expressed genes selected by subtractions were classified into six categories according to their putative functions generated by BLAST analysis. Expression of five selected genes, Met, SAM, V-ATPase, Cry (Cryptochrome gene), and An (diphthine synthase gene) were examined by RT-PCR and Real-time PCR. Both RT-PCR and Real-time PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence by exogenously applied gibberellic acid. This is the first genome-wide study of senescence in a species of turfgrass.