• 식물병연구
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• 제14권3호
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• pp.229-232
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• 2008

모자이크 증상의 과꽃(Callistephus chinensis L.)으로부터 Cucumber mosaic virus (CMV)의 한 계통(Cas-CMV)를 분리하고, Fny-CMV와 As-CMV를 대조로 기주반응, dsRNA, RT-PCR 및 RFLP분석을 통하여 바이러스를 동정하였다. Cas-CMV의 특징적인 기주반응의 차이는 박과 식물에서 발현되는 강한 병정이었으며, 특히 쥬키니호박에 접종하였을 때에는 접종 15-20일 후에 심한 모자이크 증상과 함께 어린 식물이 고사되는 괴저현상을 나타냈다. DsRNA분석과 RT-PCR실험의 결과는 Cas-CMV가 서브그룹 I의 CMV에 속하는 것으로 나타났으며, 더욱이 HindIII를 이용한 RFLP 분석은 Cas-CMV가 서브그룹 IA 구분되었다.

• Plant Resources
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• 제2권2호
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• pp.69-74
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• 1999

A rapid and sensitive assay for specific detection and identification of barley yellow mosaic virus(BaYMV) was set up using the reverse transcriptase polymerase chain reaction(RT-PCR). A couple of primers was select to discriminate the viruses. PCR fragments of BaYMV(ca.0.9 kb) were obtained by using the method designed for BaYMV capsid protein. RT-PCR fragments were cloned with vector pT7 Blue and the resulting clones were sequenced. Capsid protein of BaYMV consisted of 297 amino acids and 891 nucleotides. The capsid protein sequence of BaYMV showed that 98% of nucleotides and 99% of amino acids homology.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
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• pp.171-171
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• 2008

We monitored the occurrence of human enteric viruses in urban rivers by cell culture-PCR and RT-nested PCR. Water samples were collected monthly or semimonthly between May 2002 and March 2003 in four urban tributaries. Enteric viruses were detected by RT-nested PCR and cell culture-PCR based on a combination of Buffalo Green monkey kidney (BGMK) and A549 cell lines, followed by phylogenetic analysis of amplicons. By RT-nested PCR analysis, 45 (77.6%), 32 (55.2%), 32 (55.2%), 26 (44.8%), 12 (20.7%), 2 (3.4%), 4 (6.9%), and 4 (6.9%) of 58 samples showed positive results with adenoviruses, enteroviruses, noroviruses (NV) genogroup I (GI) and II (GII), reoviruses, hepatitis A viruses, rotaviruses and sapoviruses, respectively. Adenoviruses were most often detected and only eight (13.8%) samples were negative for adenoviruses and positive for other enteric viruses in the studied sites. Thirty-one (77.5%) of the 40 samples were positive for infectious adenoviruses and/or enteroviruses based on cell culture-PCR, and the frequency of positive samples grown on A549 and BGMK (65.0%) was higher than that grown on BGMK alone (47.5%). The occurrence of each enteric virus, except reoviruses and hepatitis A viruses was not statistically correlated with the water temperature and levels of fecal coliforms according to Binary logistic regression model. By sequence analysis, most strains of adenoviruses and enteroviruses detected in this study are similar to the causative agent of viral diseases in Korea and most NV GI- and GII-grouped strains were closely related to the reference strains from China and Japan, and GII/4-related strains had similar sequences to strains recognized as a worldwide epidemic outbreak. Our results suggested that monitoring human enteric viruses is necessary to improve microbial quality and cell culture-PCR using the combination of A549 and BGMK cells and the adenovirus detection by PCR could be useful for monitoring viral contamination in the aquatic environment.

• 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.

• 한국발생생물학회지:발생과생식
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• 제24권3호
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• pp.241-248
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• 2020

This study used a PCR-based genetic analysis platform to create a hierarchical polar dendrogram of Euclidean genetic distances for two salmonid species, Oncorhynchus mykiss (rainbow trout, RT) and Oncorhynchus masou (masu salmon, MS). The species were distantly related to other fish species based on PCR results from using the designed oligonucleotide primer series. Five oligonucleotide primers were used to generate 330 and 234 scorable fragments in the RT and MS populations, respectively. The DNA fragments ranged in size from approximately 50 bp to more than 2,000 bp. The bandsharing (BS) results showed that the RT population had a higher average BS value (0.852) than that for the MS population (0.704). The genetic distance between individuals supported the presence of adjacent affiliation in cluster I (RT 01-RT 11). The observation of a significant genetic distance between the two Oncorhynchus species verifies that this PCR-based technique can be a useful approach for individual- and population-based biological DNA investigations. The results of this type of investigation can be useful for species safekeeping and the maintenance of salmonid populations in the mountain streams of Korea.

• 미생물학회지
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• 제49권3호
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• pp.270-274
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• 2013

서양뒤영벌(Bombus terrestris)은 꿀벌의 봉군붕괴증후군(colony collapse disorder)에 대한 대체 화분매개곤충으로서 농업분야에서 중요한 역할을 하고 있다. 최근 서양뒤영벌에서 바이러스, 세균, 응애 등의 여러 병원체와 기생체가 발견되었고, 이들은 서양뒤영벌의 수명과 생식력 등에 영향을 주는 것이 알려져 있다. 서양뒤영벌 야외개체군에서 Nosema spp.를 탐지하기 위해, 서양뒤영벌 성충으로부터 genomic DNA를 추출하여 Nosema spp. 유전자들에 대해 polymerase chain reaction (PCR)을 수행하였다. 이들 유전자 중에서 small subunit ribosomal RNA (SSU rRNA) 유전자만이 증폭되었고, 염기서열분석을 통해 N. ceranae로 확인된 것은 조사된 야외개체군에서 N. ceranae가 서양뒤영벌의 주된 감염체임을 보여준다. Quantitative real-time PCR (qRT-PCR)을 이용하여 SSU rRNA 유전자를 탐지하기 위해, 먼저 PCR을 통해 SSU rRNA 유전자의 2개 영역에 대한 유전자 특이적 증폭을 확인하였다. qRT-PCR을 이용하여 각 개체에서 얻은 genomic DNA의 순차적인 농도희석를 통해 $0.85ng/{\mu}l$ 이하의 genomic DNA 농도에서도 SSU rRNA 유전자가 성공적으로 증폭되는 것이 확인되었다. 이러한 실험 결과, qRT-PCR를 이용한 N. ceranae 특이 유전자 증폭은 서양뒤영벌의 병원체 감염 진단 뿐만 아니라 생태계 위해성 평가에도 활용될 수 있을 것으로 사료된다.

• 한국수산과학회지
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• 제30권6호
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• pp.948-955
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• 1997

Immunomagnetic separation of virus coupled with .reverse transcription-polymerase chain reaction (IMS-PCR) was performed with infectious hematopoietic necrosis virus (IHNV). A DNA fragment of expected size was synthesized in the RT-PCR with total RNA extracted from IHNV inoculated CHSE-214. In a SDS-PAGE analysis, a protein band of over 70kDa was detected from non-infected cells and cells inoculated with IHNV and infectious pancreatic necrosis virus (IPNV). This protein was detected in the Western blot analysis probably because of non-specific reaction to monoclonal antibody against IHNV nucleocapsid protein. In the immunomagnetic separation, magnetic beads coated with monoclonal antibody against the IHNV nucleocapsid protein was incubated with supernatant from IHNV inoculated CHSE-214 cells. During this process, the non-specifically reacting protein could be removed by washing the magnetic bead with PBS in the presence of an external magnetic field, and viral proteins were detected from the remaining, cleaned magnetic beads. It was necessary to extract viral RNA from the captured virus particles before RT-PCR, and no DNA product was detected when the captured virus was only heated 5 min at $95^{\circ}C$. A PCR-product of expected size was synthesized from IMS-PCR with magnetic beads double coated either by goat anti-mouse IgG antibody -monoclonal antibody or streptavidin - biotin conjugated monoclonal antibody.

• 한국식물병리학회지
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• 제14권4호
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• pp.314-318
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• 1998

Using the reverse transcription polymerase chain reaction (RT-PCR), a rapid and sensitive assay method for the detection and identification of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) was adapted. Two units of primers from each virus were selected and used for the determination of two different viruses. PCR fragments of BaYMV (ca. 0.9kb) and BaMMV (ca. 0.8kb) were obtained from the designed method for the assay of BaYMV and BaMMV coat protein. PT-PCR fragments were cloned using vector pT7 Blue and the sequences of the selected clones were analyzed. coat protein of BaYMV and that of BaMMV consisted of 297 amino acids (891 nucleotides) and 251 amino acids (753 nucleotides), respectively. The snalysis of coat protein genes from these two viruses showed that 45.6% of nucleotides sequence ad 34.9% of amino acid in BaYMV were homologous to those in BaMMV.

• 한국식품과학회지
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• 제44권5호
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• pp.638-642
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• 2012

돼지축사에서 채집해온 6개의 육성돈의 분변에서 식중독 유발 바이러스인 HAV와 HEV를 검출하였으며, HAV는 88.3%의 검출율을 보였으며, HEV는 33.3%의 검출율을 보였다. 결과에는 제시하지 않았으나, 염기서열 분석결과 HEV는 사람에게 전염이 가능한 유전자형인 III형이었으며, 실험적으로 사람의 간세포인 PLC/PRF/5에 접종하였을 때 증식이 됨을 확인하였다. 식중독 유발 바이러스인 HAV와 HEV는 오염된 식품이나 물을 섭취하거나 교차 오염에 의해 전염이 가능하기 때문에 돼지축사에서 위생상태의 개선뿐만 아니라 육류를 섭취하기 전인 운송 및 가공과정까지 식중독 유발 바이러스에 의한 교차오염을 막는 노력이 필요하다. HAV와 HEV 모두 검출된 분변에서 HAV를 순수분리하고 빠르게 검출하기 위해 IMS-RT-PCR을 적용하였으며, 항원-항체 반응에 의해 순수하게 HAV만을 분리할 수 있었다. 또한 HAV만이 순수분리 되었는지 재확인하기위해 세포감염을 통해 증식된 바이러스를 확보한 후 nested RT-PCR을 수행한 결과, HAV만을 순수 분리할 수 있음을 확인하였다. 이는 IMS 활용기술이 단순히 항체를 교체함으로써 다른 특정 식중독 유발 바이러스의 다양한 시료에서 바이러스 순수분리 및 검출에 활용 가능성이 있음을 확인하였다.

• 한국자원식물학회지
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• 제21권3호
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• pp.210-215
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• 2008

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer $Express^{TM}$ software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.