• 미생물학회지
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• 제55권1호
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• pp.25-32
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• 2019

Influenza A (H1N1) virus caused a worldwide pandemic in 2009-2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

• Journal of Microbiology and Biotechnology
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• 제21권1호
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• pp.100-108
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• 2011

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

• 한국미생물·생명공학회지
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• 제36권1호
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• pp.34-42
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• 2008

소의 혈액, 세포, 조직, 기관 등 소 유래 물질을 원료로 사용한 생물의약품, 조직공학제제, 세포치료제의 경우 소 유래원료 물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. BVDV는 동물세포주, 우혈청 등에 가장 흔하게 오염되는 바이러스이다. 소 유래물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BVDV안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BVDV를 정량적으로 검출하고, 제조공정에서 BVDV제거 검증을 위한 시험법으로 활용이 가능한 BVDV real-time RT-PCR시험법을 확립하였다. BVDV에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BVDV RNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 $1TCID_{50}/mL$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성 (specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BVDV를 오염시킨 CHO 세포주와 소 유래콜라겐에서 BVDV검출 시험을 실시하였다. BVDV를 감염시킨 CHO 세포와 세포배양 상청액에서 BVDV를 정량적으로 검출할 수 있었다. 소 유래 콜라겐에서도 $10TCID_{50}/mL$까지 정량적으로 검출할 수 있었다.

• 미생물학회지
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• 제44권3호
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• pp.228-236
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• 2008

세포배양 유래 생물의 약품 생산 공정에서 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. Reovirus type 3 (Reo-3)는 동물 세포주와 동물 세포 배양 공정에 오염되는 대표적인 바이러스이다. 세포배양 유래 생물의약품의 Reo-3 안전성을 확보하기 위해, 세포주, 원료물질, 제조공정, 완제품에서 Reo-3를 정략적으로 검출하고, 제조공정에서 Reo-3 제거 검증을 위한 시험법으로 활용이 가능한 real-time RT-PCR 시험법을 확립하였다. Reo-3에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 Reo-3 RNA 정략 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 $3.2{\times}10^0\;TCID_{50}/ml$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성(specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 Reo-3를 오염시킨 CHO 세포에서 Reo-3 검출 시험을 실시한 결과 Reo-3를 감염시킨 CHO 세포와 세포배양 상청액에서 Reo-3를 정략적으로 검출할 수 있었다. 또한 바이러스필터 공정에서 Reo-3제거 효과를 감염역가 시험법과 비교 검증한 결과 더 빠른 시간에 동일한 결과를 얻을 수 있었다. 위와 같은 결과에서 확립된 Reo-3 real-time RT-PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의 약품 생산 공정 검증, 바이러스 제거 공정 검증 등에서 감염 역가 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다.

• KSBB Journal
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• 제23권4호
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• pp.303-310
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• 2008

소의 혈액, 세포, 조직, 기관 등 소유래 물질을 원료로 사용한 생물의약품, 조직공학제제, 세포치료제의 경우 소유래 원료물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. BPIV3는 동물 세포주, 우혈청 등에 가장 흔하게 오염되는 바이러스이다. 소유래 물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BPIV3 안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BPIV3를 정량적으로 검출하고, 제조공정에서 BPIV3 제거 검증을 위한 시험법으로 활용이 가능한 BPIV3 real-time RT-PCR 시험법을 확립하였다. BPIV3에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BPIV3 RNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 2.8 $TCID_{50}/mL$이었다. 확립된 시험법의 신뢰성 (reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성 (specificity)과 재현성 (reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의 약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BPIV3를 오염시킨 CHO 세포주와 소유래 콜라겐에서 BPIV3 검출 시험을 실시하였다. BPIV3를 감염시킨 CHO 세포와 세포배양 상청액에서 BPIV3를 정량적으로 검출할 수 있었다. 소유래 콜라겐에서도 7.8 $TCID_{50}/mL$ 까지 정량적으로 검출할 수 있었다. 위와 같은 결과에서 확립된 BPIV3 real-time RT-PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의약품 생산 공정 검증, 바이러스 제거 공정 검증 등에서 감염역가 시험법과 같은 생물학적 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.132-137
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• 2008

Differential gene expression profiling was carried out in the hepatic tissue of medaka fish, Oryzias latipes, after exposure to an organophosphorus pesticide (OPP), Iprobenfos (IBP), a widely used pesticide in agri- and fish-culture, using a medaka cDNA micro array. Twenty six kinds of differentially expressed candidate genes, with 15 and 11 induced and repressed in their gene expressions, respectively, were associated with cytoskeleton (3.8%), development (7.7%), immune (7.7%), metabolism (30.8%), nucleic acid/protein binding (42.3%) and reproduction (7.7%). Of these genes, changes at the transcription level of five were re-evaluated by real-time quantitative PCR (qRT-PCR). Considering the known function of authentic genes, the effects of IBP on the biological activity and pathological aspects in medaka fish were discussed. The identified genes could be used as molecular biomarkers for biological responses to OPPs contamination in an aquatic environment.

• International Journal of Oral Biology
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• 제30권3호
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• pp.99-103
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• 2005

Bone morphogenetic proteins (BMPs), members of a large group of TGF-beta family, are important molecular regulators of morphogenesis of numerous tissues and organs, including bones and teeth. Most BMPs are capable of inducing bone formation in vivo and therefore are of considerable clinical interest for regenerating mineralized tissues. Recently, we have developed a method to culture cells from human cementum (human cementum-derived cells, HCDCs). HCDCs, when attached to synthetic hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic and transplanted into immunodeficient mice, formed histologically identifiable cementum-like tissue. Since it is unclear to what extent BMPs are involved in cementogenesis, the aim of this study was to establish which BMPs are expressed by cementogenic HCDCs and whether the expression of BMPs is related to the degree of cellular differentiation in vitro. HCDCs were maintained in growth medium (DMEM/F12 supplemented with 10% FBS) until confluent (proliferation stage). Upon reaching confluence, cells were incubated in the differentiation medium (DMEM/F12 medium containing 10% FBS and 50 mg/ml ascorbic acid) for 14 days (differentiation stage). Next, HCDCs were incubated in mineralization medium (DMEM/F12, 50 mg/ml ascorbic acid, 2.5 mg/ml of ITS (insulin-transferrinselenium), 5 mM beta-glycerophosphate and $10^{-8}M$ dexamethasone) for another 14 days (mineralization stage). At the end of each differentiation stage, total RNA was isolated and evaluated for BMPs (2 through 8) expression by employing real time RT-PCR. HCDCs expressed most of BMPs examined except BMP-7 and BMP-8. Furthermore, on average, the highest levels of BMPs were expressed at the earlier differentiation stage, prior to the initiation of mineralization in vitro. These results indicate that several BMPs are expressed during cementoblastic differentiation and suggest that BMPs may be involved in the homeostasis of human cementum.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.267-272
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• 2013

Chicken-type lysozyme (c-lysozyme) is present in shrimp and is active against some bacteria. To further understand the regulation of c-lysozyme in the fleshy shrimp Fenneropenaeus chinensis, we determined the tissue-specific gene expression of c-lysozyme and the time-course of mRNA expression in response to Vibrio anguillarum and lipopolysaccharide (LPS) injection by quantitative reverse real-time polymerase chain reaction. The results showed that c-lysozyme was expressed in all tissues tested, including gill, eyestalk, eye, hemocytes, hepatopancreas, intestine, heart, and pleopod. It was most highly expressed in the intestine followed by the eyestalk, gill, hemocytes and hepatopancreas. The mRNA expression level began to decline in a short time after V. anguillarum challenge and was then upregulated by two fold or more at 24 h post injection (hpi) compared to that at 0 h. Expression was suppressed shortly after LPS injection and began to increase with higher levels of 5.8-, 5.2- and 8.4-fold at 24, 48, and 72 hpi, respectively. Higher expression was sustained and showed a gradual increasing trend until the end of the experiment (72 hpi). These results increase our understanding of the regulation of defense mechanisms and facilitate an evaluation of the effects of probiotics or immunostimulants in shrimp culture.