• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.838-843
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• 2005

Brain-derived neurotrophic factor (BDNF) is a small secretory protein and a member of the nerve growth factor (NGF) gene family. We cloned the flounder BDNF gene from a flounder brain cDNA library. The nucleotide sequence of the cloned gene showed an open reading frame (ORF) consisting of 810 bp, corresponding to 269 amino acid residues. The tissue distribution of flounder BDNF was determined by reverse transcription-polymerase chain reaction (RT-PCR) in brain, embryo, and muscle tissues. To express fBDNF using a eukaryotic expression system, we constructed the vector mpCTV-BDNF containing the fBDNF gene and transformed this vector into Chlorella ellipsoidea. Stable integration of introduced DNA was confirmed by PCR analysis of genomic DNA, and mRNA expression in C. ellipsoidae was confirmed by RT-PCR analysis.

• 환경생물
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• 제22권2호
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• Journal of Veterinary Science
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• 제21권2호
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• pp.22.1-22.9
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• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• 식물병연구
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• 제16권2호
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• pp.121-124
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• 2010

박과작물에 발생하는 종자전염 바이러스 3종(CGMMV, KGMMV, ZGMMV)에 대한 검출을 위해 IC-RT-PCR의 적용 가능성을 시험하였다. IC-RT-PCR을 이용하여 감염 종자와 잎으로 부터 바이러스의 특이적인 검출이 가능하였으며, 검출 민감도는 ELISA 보다 100배 이상 높았다. 또한 반응을 마친 ELISA 마이크로플레이트에 남아있는 항원을 IC-RT-PCR의 template로 사용할 경우 ELISA 결과를 곧바로 확인할 수 있었다. 따라서 IC-RT-PCR에 의한 본 검정방법은 박과작물의 대규모 포장검사 및 종자검사에 편리하게 사용될 수 있을 것으로 기대된다.

• 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.

• Pediatric Infection and Vaccine
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• 제30권2호
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• pp.62-72
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• 2023

목적: Coronavirus disease 2019 (COVID-19) 발생 이후 인간 코로나바이러스를 포함한 호흡기 바이러스의 패턴에 변화가 있을 것으로 생각되어, COVID-19 발생 이전 경기도 명지병원에 급성 호흡기 감염증으로 입원한 소아청소년을 대상으로 바이러스 감염의 패턴을 보고하였다. 방법: 2017년 1월부터 2019년 12월까지 급성 호흡기 감염증으로 명지병원에 입원한 18세 이하 소아청소년을 대상으로 하였고 real-time reverse transcription polymerase chain reaction (RT-PCR) 결과를 바탕으로 후향적으로 의무기록을 고찰하였다. 결과: 총 3,557명 중에서 중복감염 포함하여 3,686건의 바이러스가 검출되었고 평균 양성률은 78.6%이었으며 여름에 비해 겨울에 PCR 양성률이 높았다. 파라인플루엔자바이러스, 메타뉴모바이러스, 보카바이러스는 4-6월에 주로 검출되고, 인간엔테로바이러스는 7-9월에, 호흡기세포융합바이러스와 인플루엔자바이러스는 겨울철에 주로 검출되었다. 진단별로 인두편도염에서는 아데노바이러스가 가장 많았고 폐렴에서는 호흡기세포융합바이러스가 가장 많이 검출되었다. 인간 코로나 바이러스는 겨울철에 주로 검출되었으며, 크루프 환자 중에서 3번째 높은 빈도로 나타났다. 결론: 본 연구결과가 이전에 보고되었던 소아청소년의 호흡기 바이러스 역학과 크게 다르지 않았다. 또한 COVID-19 발생 이후 호흡기 바이러스 패턴 비교 연구에 도움이 되고자 COVID-19 발생 이전 호흡기 바이러스 역학정보를 보고하는 바이다.

• 대한바이러스학회지
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• 제27권1호
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• 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.

• 대한소아치과학회지
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• 제42권4호
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• pp.312-318
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• 2015

본 연구의 목적은 치과분야에서 줄기세포 공급원으로써 과잉치의 활용 가능성을 알아보고자 Real Time Quantitative Reverse Transcription Polymerase Chain Reaction (Real Time qRT-PCR)법을 이용하여 발치된 과잉치 치수 유래 줄기세포 (supernumerary dental pulp stem cells, sDPSCs)로부터 상아모세포로의 분화여부를 관찰해 보는 것이었다. 이를 위해 상아모세포의 대표적인 발현인자로 알려진 alkaline phosphatase (ALP), osteocalcin (OC), osteonectin (ON), dentin matrix acidic phosphoprotein 1 (DMP-1) 그리고 dentin sialophosphoprotein(DSPP)의 발현을 분화제 처리 후 0일, 8일 그리고 14일째에 각각 Real Time qRT-PCR 법을 통해 상대적인 mRNA의 발현 양을 비교하여 변화 양상을 알아보았다. 또한 Alizarin-red solution 의 염색을 통해 sDPSCs가 분화제 처리 7일, 14일, 21일 그리고 28일째에 석회화 결절을 형성하는 정도를 시각적으로 확인해 보았다. Real Time qRT-PCR 결과 분화제 처리 8일째에 가장 높은 발현 양을 보이다가 14일째에 감소하는 추세를 나타내었으며, Alizarin-red solution 염색 결과는 7일째부터 흐리게 나타나다가 14일째에는 배지 전반에 걸쳐 진하게 염색되는 소견을 보였다. 따라서, sDPSCs를 이용한 연구에서 Real Time qRT-PCR법을 위한 분화제의 처리 기간은 8일 정도가 적절하며, Alizarin-red solution 염색은 14일이 적당한 것으로 사료된다.

• Journal of Veterinary Science
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• 제23권6호
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• pp.80.1-80.6
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• 2022

A 10-year-old male neutered Labrador Retriever presented with a history of acute-onset tachypnoea, lethargy and anorexia. The dog was pyrexic, tachypnoeic and dyspnoeic on examination. A rapid antigen test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was performed on an oropharyngeal swab and yielded a positive result. SARS-CoV-2 infection was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis. Both of the dog's owners had positive rapid antigen test and RT-PCR analysis results for SARS-CoV-2. Additional diagnostics included computed tomography. Resolution of the dog's clinical signs was achieved with symptomatic treatment.