• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.881-884
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• 1999

The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide-incorporation activity on an in vitro transcribed RNA containing the 3' end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.

• Journal of Plant Biology
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• 제38권3호
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• pp.267-274
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• 1995

Based upon the nucleotide sequence of As strain of cucumber mosaic virus (CMV-As0 RNA4, coat protein (CP) gene was selected for the design of oligonucleotide primers of polymerase chain reaction (PCR) for detection and identification of the virus. Reverse transcription and polymerase chain reaction (RT-PCR) was performed with a set of 18-mer CMV CP-specific primers to amplify a 671 bp fragment from crude nucleic acid extracts of virus-infected leaf tissues as well as purified viral RNAs. The minimum concentrations of template viral RNA and crude nucleic acids from infected tobacco tissue required to detect the virus were 1.0 fg and 1:65,536 (w/v), respectively. No PCR product was obtained when potato virus Y-VN RNA or extracts of healthy plants were used as templates in RT-PCR using the same primers. The RT-PCR detected CMV-Y strain as well as CMV-As strain. Restriction analysis of the two individual PCR amplified DNA fragments from CMV-As and CMV-Y strains showed distinct polymorphic patterns. PCR product from CMV-As has a single recognition site for EcoRI and EcoRV, respectively, and the product from CMV-Y has no site for EcoRI or EcoRV but only one site for HindIII. The RT-PCR was able to detect the virus in the tissues of infected pepper, tomato and Chinese cabbage plants.

• 식물병연구
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• 제11권2호
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• pp.98-105
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• 2005

기주 식물체 내에서 식물바이러스의 증식과 이동 여부는 바이러스 게놈과 기주 간의 상호작용에 의해 결정된다. 바이러스는 기주 내에서 바이러스가 증식하고 이동하기 위해서는 기주의 요소들을 이용해야 하며, 이러한 기주 요소들은 바이러스의 기주내 침입(entry),바이러스 유전자의 발현, 그리고 바이러스 입자형성(virion assembly) 등 모든 과정에서 직접적으로 관여를 하거나, 또는 기주 단백질 발현과 저항성을 조절하여 바이러스 증식에 간접적으로 관여를 한다. 기주 요소들과 상호작용을 통해서 바이러스 증식에 관여함으로써, 기주 특이성 및 바이러스의 병 발생에 관여를 할 것으로 보고 있다.

• Korean Chemical Engineering Research
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• 제46권4호
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• pp.647-659
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• 2008

콜로이드는 거시적으로 균일한 성질을 갖는 입자분산계이다. 콜로이드 입자는 다양한 입자분산계의 모델로서 많은 기초연구가 이루어져 왔을 뿐만 아니라, 산업적으로 다양하게 응용이 되었다. 최근에는 나노-바이오 관련 연구에 적용되어 새롭게 각광을 받고 있는 나노 소재중 하나이다. 본 총설에서는 입자 분산계의 정의 및 분류에 대해 간략히 기술하고, 나노-바이오 응용을 위한 표면 성질 및 표면 개질방법에 대해 다룰 것이다. 또한, 기존의 구형의 입자분산계에서 더 나아가, 모양과 크기가 제어된 입자 분산계의 합성에 관한 최근 결과를 소개하였다. 마지막으로, 콜로이드 입자의 나노-바이오 응용분야로서, 금속 콜로이드 잉크와, 3차원 콜로이드 결정을 활용한 나노-바이오 센서, 및 2차원 콜로이드 구조를 이용한 패턴제작과 응용 연구에 대해 살펴보았다.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.358-367
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• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

• 미생물학회지
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• 제47권1호
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• pp.30-37
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• 2011

조류 인플루엔자 바이러스(AIV) 아형을 ultra-time PCR법(UPCR)을 이용하여 초스피드로 진단할 수 있는 방법을 고안하였다. 표적 대상의 프라이머는 AIV H5N1 아형의 hemagglutinin(HA) 유전자 중 가장 상보성이 높은 133 bp의 부위를 선택하였고, 실험의 안전을 위하여 인공합성의 방법으로 제작하였다. 압타머와 결합한 molecular beacon 기반 Mini-Opticon Q-PCR 기기를 사용한 UPCR법으로, 총 UPCR 반응액의 양을 10 ${\mu}l$으로, UPCR과 용융온도 분석시간을 15분 이내로 매우 짧게 단축시켰다. 민감도 측정에서 최소의 주형인 5분자의 HA 유전자만으로 정확히 AIV의 특이적 133 bp를 합성하였다. UPCR로 디자인된 이 PCR은 AIV 아형의 진단에 적용될 수 있을 뿐 아니라, UPCR이 기반되는 진단을 이용하여 다른 병원체에도 널리 적용 될 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• 대한수의학회지
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• 제47권4호
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• pp.399-407
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• 2007

Cause of high lethality and dissemination to human being, new development of rapid method for the detection of highly pathogenic Avian Influenza Virus (AIV) is still necessary. For the detection of AIV subtype H5N1, typical pathogenic AIV, new method to confirm sub-typing of this virus is also needed. For the purpose of ultra-rapid detection and sub-typing of hemagglutinin and neuraminidase of AIV, this study was planned. As the results we could demonstrate an ultra-rapid multiplex real-time PCR (URMRT PCR) for the detection of AIV In this study, the URMRT PCR were optimized with synthesized AIV H5- and AIV Nl-specific DNA templates and GenSpector TMC, which is a semiconductor process technology based real-time PCR system with high frequencies of temperature monitoring. Under eight minutes, the amplifications of two AIV subtype-specific PCR products were successfully and independently detected by 30 cycled ultra-rapid PCR, including melting point analysis, from $1{\times}10^3$ copies of mixed template DNA. The URMRT PCR for the detection of AIV H5N 1 developed in this study could be expected to apply not only detections of different AIVs, but also various pathogens. It was also discussed that this kind of the fastest PCR based detection method could be improved by advance of related technology in near future.

• 한국동물위생학회지
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• 제37권3호
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• pp.219-224
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• 2014

A eight-month-old blue and yellow macaw (Ara ararauna) with psittacine beak and feather disease (PBFD)-suspected signs, such as, abnormal feather, depression and diarrhea, was presented to Animal Disease Intervention Center, Kyungpook National University in 16 April 2014. The partial ORF V1 gene of PBFD virus (PBFDV) was detected by polymerase chain reaction (PCR) from DNA templates extracted from feather, blood and cloacal swab sample of the bird, but no other viral DNAs that often infected in psittacine birds including avian bornavirus and avian polyomavirus were detected from the samples of the bird, indicating this case is due to single infection of PBFDV. Nucleotide sequence analysis of the amplified partial ORF V1 gene was confirmed to have 96.7% and 93.6% homology with that of previously reported PBFDV strain (Genbank no. HM748924 and FJ685980). This report describes the first detection of PBFDV in PBFD-suspected blue and yellow macaw in Korea.

• 한국양식학회지
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• 제10권2호
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• pp.171-178
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• 1997

Infectious pancreatic necrosis virus (IPNV)는 치어에 감염되어 치명적인 질병을 유발하는, 양식산업에 있어 중요한 어류 병원체이다. 본 연구에서는 IPNV를 신속, 정확하게 진단하는 방법을 개발하고자 IPNV의 항원성 단백질인 VP2 유전자 부분에서 선택한 primers를 이용하여 역전사-중합효소연쇄반응법(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)을 실시하였다. RT-PCR 증폭법으로 순수분리도니 IPNV dsRNA 40 ng 정도의 적은 양도 확인 할 수 있었으며, IHNV와 같은 다른 어류 병원체의 게놈을 RT-PCR templates로 사용하였을 경우는 어떠한 PCR 산물도 검출되지 않는 특이성을 보였다. 특히 유전자의 분리없이 조직 그 자체를 대상으로 RT-PCR을 행하는 in situ RT-PCR 방법으로 IPNV가 감염된 넙치 (Paralichthy olivaceus) 치어의 조직에서 IPNV 감염을 신속하게 확인할 수 있었다. 따라서 RT-PCR 및 in situ RT-PCR 방법은 IPNV를 신속, 정확하게 진단하는데 활용될 수 있을 것으로 생각된다.