• 한국산림과학회지
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• 제96권4호
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• pp.425-431
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• 2007

Rhizina undulata의 PCR 검정 및 유전적 특성 분석을 목적으로, rDNA ITS 영역의 염기배열 해석 및 PCR 방법에 의한 토양으로부터 R. undulata의 진단법을 개발하였다. 18S rDNA 부분의 염기서열 분석 결과, 공시한 4종의 균주 모두 1,375 nt의 크기로 동일하였으며, 염기배열도 100% 일치하였다. 한편, rDNA ITS 영역의 염기배열은 585 nt이었고, PDK-1, PTT-1 및 PDJ-9 균주는 염기배열이 100% 동일하였으나, PDS-5균주에서는 두 곳에서 염기의 치환이 발견되었다. 이와 같은 염기배열을 분석하여 제작한 R. undulata rDNA ITS 영역 특이적 primer를 이용한 PCR 검정 결과, R. undulata 균주들에서만 약 525 bp 크기의 ITS 영역 특이적인 증폭산물이 검출되었다. PCR 방법에 의하여 검출할 수 있는 토양 중의 R. undulata 최소 균사량의 한계를 확인하기 위해서, 순수 배양한 R. undulata 균사현탁액을 순차 희석하여 100g의 사양토에 혼합한 다음, 농도별로 균사 혼합한 각각의 토양 시료로부터 추출한 total DNA의 PCR 증폭산물을 분석한 결과, PCR 방법에 의하여 100g의 토양 중에 1 ng의 R. undulata 균사가 함유되어 있는 경우까지 검출이 가능하였다.

• Bulletin of the Korean Chemical Society
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• 제30권12호
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• pp.2999-3005
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• 2009

Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using the early gene product, E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using gene recombinant technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwichtype binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 commonly used for the cell lysis. Temperature also affected the stability of the E7 protein, and we found that the E7 protein was stabilized at 4$^{\circ}C$ for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid test could specifically detect HPV-infected samples.

• Bulletin of the Korean Chemical Society
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• 제30권12호
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• pp.2993-2998
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• 2009

An enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor combined with cell concentration technology based on immuno-magnetic separation (IMS) was investigated for use as a potential tool for early screening of Listeria monocytogenes (L. monocytogenes) in food products. The target analyte is a well-known pathogenic foodborne microorganism and outbreaks of the food poisoning typically occur due to contamination of normal food products. Thus, the aim of this study was to develop a rapid and reliable sensor that could be utilized on a daily basis to test food products for the presence of this pathogenic microorganism. The sensor was optimized to provide a high detection capability (e.g., 5.9 ${\times}\;10^3$ cells/mL) and, to eventually minimize cultivation time. The cell density was condensed using IMS prior to analysis. Since the concentration rate of IMS was greater than 100-fold, this combination resulted in a detection limit of 54 cells/mL. The EOC-IMS coupled analytical system was then applied to a real sample test of fish intestines. The system was able to detect L. monocytogenes at a concentration of 2.4 CFU/g after pre-enrichment for 6 h from the onset of cell cultivation. This may allow us to monitor the target analyte at a concentration less than 1 CFU/g within a 9 h-cultivation provided a doubling time of 40 min is typically maintained. Based on this estimation, the EOC-IMS system can screen and detect the presence of this microorganism in food products almost within working hours.

• 대한화학회지
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• 제54권2호
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• pp.215-221
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• 2010

살모넬라는 음식과 식수에서 흔히 나오는 중요한 병원체로 세계 곳곳에서 급성 위장염과 같은 감염증을 일으키며, 일반적으로 인간의 혈청형 임상종으로는 Salmonella enterica의 혈청형인 S. Typhimurium과 S. Enteritidis가 있다. 일반적인 검출 방법으로 살모넬라를 기본으로 하여 선택적인 배양으로 샘플을 수집하였고 살모넬라를 일으키는 군체의 특징을 생화학과 혈청학상인 테스트를 하였으나 이러한 방법들은 일반적으로 시간이 걸리고 높은 감도를 보이지 않았다. 최근, 등온증폭반응법과 real-time PCR법을 이용하여 높은 감도, 특이성으로 현재 병원성 박테리아에 빠르게 수행할 수 있게 되었다. 본 연구에서는 등온증폭반응과 real-time PCR법을 사용하여 S. Typhimurium과 S. Enteritidis의 검출하였다. 선택적인 타겟 유전자로, invA를 살모넬라종의 염기서열에 특이적으로 임의복제 하였다. 등온증폭반응과 real-time PCR은 살모넬라종으로부터 임의의 염기서열을 증폭하여 검출하였고, invA는 S. Typhimurium과 S. Enteritidis의 두 가지 종을 모두 검출하였다. 이러한 등온증폭반응과 real-time PCR법으로 S.Typhimurium과 S. Enteritidis의 검출 가능성을 보였으며, 살모넬라 종에 대한 특이성, 민감성을 갖춘 유용한 검출방법을 제시하였다.

• 생명과학회지
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• 제27권12호
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• pp.1393-1402
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• 2017

식물에서, 칼슘-의존적 단백질 카이네즈(CDPKs)는 $Ca^{2+}$ 신호전달에서 중요한 $Ca^{2+}$ 수용체이다. 벼(Oryza sativa L.)의 CDPKs인 3개의 OsCPKs는 생물정보에 대한 분석이 이루어졌으나, OsCPK11 유전자는 연구가 완전히 수행되지 않았다. 다양한 조직에서 OsCPK11 유전자가 전사수준에서 발현한다는 것은 알려져 있으나, 단백질 수준에서 발현과 생화학적인 특징은 잘 알려져 있지 않다. 이 연구는 OsCPK11의 몇 가지 생화학적 특징을 알아보기 위해 이루어졌다. 먼저 in vitro에서 E. coli를 이용하여 GST-OsCPK11를 발현시키고, 카이네즈 활성 측정과 칼슘-의존적 단백질 카이네즈로서 OsCPK11의 생화학적 분석도 수행하였다. OsCPK11은 스스로 자가인산화하며, $Ca^{2+}$의 존재 하에서 기질로서 histone III-s와 MBP로 인산기 전달 작용을 수행한다. 재조합 OsCPK11의 활성은 $Mg^{2+}$에 의해 영향을 받으며, pH 7.0-7.5에서 최적의 활성을 보인다. 또한 OsCPK11의 활성은 높은 수준의 $Ca^{2+}$가 존재하는 조건에서는 $Mg^{2+}$, $Mn^{2+}$, $Na^+$의 영향을 받지 않는다. 또한 OsCPK11의 자가인산화는 OsCPK11의 $Ca^{2+}$ 민감도를 감소시키는 것으로 밝혀졌다. 마지막으로, OsCPK11의 N-말단 다양화 지역으로 토끼 항체를 만들었고, immunoblot을 기초로 polyclonal antibody는 95.5 kD의 GST-OsCPK11를 인식하는 것으로 나타났다. 이 결과는 벼의 $Ca^{2+}$ 매개 신호전달에서 OsCPK11의 기능을 더 잘 이해하는데 도움을 줄 것이며, 심화 연구를 위해 다양한 OsCPKs의 단백질 정보를 결정하는 것이 필요할 것이다.

• 식물병연구
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• 제21권2호
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• pp.74-81
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• 2015

박과 작물에서 과일썩음병(bacterial fruit blotch)을 일으키는 Acidovorax citrulli를 종자로부터 검출하기 위한 특이적이고 민감한 nested-PCR 방법을 개발하였다. 본 연구에서는 Next Generation Sequencing을 이용하여 draft genome sequencing을 얻은 후 이를 분석하여 PCR 프라이머를 디자인하였고, 이들 프라이머의 A. citrulli에 대한 특이성을 확인하여 Ac-ORF 21F/Ac-ORF 21R의 nested PCR 프라이머를 최종 선발하였다. Ac-ORF 21F/Ac-ORF 21R는 오직 A. citrulli에서만 특이적으로 140bp 크기의 DNA를 증폭하였으며, 그 검출민감도는 1차 PCR 검출한계(10 ng genomic DNA/PCR)보다 검출한계를 10,000배 증가시켰다. 개발된 nested-PCR 방법을 통해 병원균을 인공접종한 수박 종자의 외부검사에서 $10^1cfu/ml$까지 인공 접종 한 모든 종자 시료에서 병원균을 검출하였고, 병원균을 인공접종 한 수박 종자의 내부검사에서는 병원균이 검출되지 않았다. 자연 감염 수박 종자의 외부검사에서는 10개의 반복 시료 중 2개에서, 그리고 종자 내부검사에서는 10개의 반복 시료 중 5개에서 A. citrulli를 검출하였다. 본 연구에서 개발한 nested-PCR은 특이성과 민감도가 높고 인공접종과 자연감염 수박 종자에서도 병원균의 검출이 가능하여 박과 작물의 종자로부터 A. citrulli를 검출하는데 효과적으로 사용될 수 있을 것으로 생각된다.

• 대한수의학회지
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• 제42권1호
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• pp.1-6
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• 2002

The frequencies of gamma-ray-induced micronuclei (MN) in cytokinesis-blocked (CB) lymphocytes at several doses were measured in three donors of three species (human, rabbit, dog). Measurements performed after irradiation showed a dose-related increases in MN frequency in each of the donors studied. When analysed by linear-quadratic model the line of best fit was : human : $y=0.1184D+0.01867D^2+0.01$, rabbit : $y=0.0387D+0.00528D^2+0.01$ (y = number of MN/CB cells and D = irradiation dose in Gy). The relative sensitivity of rabbit lymphocytes compared with human lymphocytes was estimated by best fitting linear-quadratic model based on the radiation-induced MN data over the range from 0 Gy to 4 Gy. In the case of MN frequency with 0.2, the relative sensitivities of rabbit lymphocytes was 0.39. These data indicate that the induction of MN in rabbit CB cells following irradiation was much less sensitive to the MN induction effects of gamma-irradiation than those from human. The MN assay with dog lymphocytes was very difficult and time-consumed because the dog PHA-stimulated lymphocytes yielded cultures with very low level of CB cells formation in the condition of this experiment. Our in vitro radiobiological study confirmed that the cytogenetic response obtained in blood from rabbit can be utilized for application in environmental studies.

• 식물병연구
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• 제12권2호
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• pp.144-147
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• 2006

닭을 이용한 egg yolk immunoglobulin (IgY)의 생산과 이용 가능성을 조사하기 위하여 pepper mild mottle virus (PMMoV)로 닭을 면역하였다. Ring-test로 달걀의 난황에서 분리 정제한 IgY의 역가를 조사한 결과 IgY의 최고 역가는 1:2,560이었고 달걀 l 개에서 대략 $60{\sim}80 mg$의 IgY를 얻을 수 있었다. 최고역가를 갖는 IgY를 이용하여 Gelrite gel 이중확산법, 효소면역항체법 (ELISA), 조직자국면역반응법을 실시한 결과 간접면역효소항체법의 PMMoV 검출 감도는 1 ng/ml 이었고, 검정법의 바이러스 검출감도와 특이도는 IgG와 비슷한 경향을 보였다. 그러므로 한 마리의 닭으로부터 대량으로 생산 가능한 IgY는 식물바이러스에 대한 항체생산과 혈청학적 검정에 유용할 것으로 생각한다.

• 대한유전성대사질환학회지
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• 제3권1호
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• pp.86-93
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• 2003

Background : The SCL began screening of newborns and high risk group blood spots with tandem mass spectrometry (MS/MS) in April 2001. Our goal was to determine approximate prevalence of metabolic disorders, optimization of decision criteria for estimation of preventive effect with early diagnosis. This report describes the ongoing effort to identify more than 30 metabolic disorders by MS/MS in South Korea. Methods : Blood spot was collected from day 2 to 30 (mostly from day 2 to 10) after birth for newborn. Blood spot of high risk group was from the pediatric patients in NICU, developmental delay, mental retardation, strong family history of metabolic disorders. One punch (3.2 mm ID) of dried blood spots was extracted with $150{\mu}L$ of methanol containing isotopically labelled amino acids (AA) and acylcarnitines (AC) internal standards. Butanolic HCl was added and incubated at $65^{\circ}C$ for 15 min. The butylated extract was introduced into the inlet of MS/MS. Neutral loss of m/z 102 and parent ion mode of m/z 85 were set for the analyses of AA and AC, respectively. Diagnosis was confirmed by repeating acylcarnitine profile, urine organic acid and plasma amino acid analysis, direct enzyme assay, or molecular testing. Results : Approximately 31,000 neonates and children were screened and the estimated prevalence (newborn/high risk group), sensitivity, specificity and recall rate amounted to 1:2384/1:2066, 96.55%, 99.98%, and 0.73%, respectively. Confirmed 28 (0.09%) multiple metabolic disorders (newborn/high risk) were as follows; 13 amino acid disorders [classical PKU (3/4), BH4 deficient-hyperphenylalaninemia (0/1), Citrullinemia (1/0), Homocystinuria (0/2), Hypermethioninemia (0/1), Tyrosinemia (1/0)], 8 organic acidurias [Propionic aciduria (2/1), Methylmalonic aciduria (0/1), Isovaleric aciduria (1/1), 3-methylcrotonylglycineuria (1/0), Glutaric aciduria type1 (1/0)], 7 fatty acid oxidation disorders [LCHAD def. (2/2), Mitochondrial TFP def. (0/1), VLCAD def. (1/0), LC3KT def. (0/1). Conclnsion : The relatively normal development of 10 patients with metabolic disorders among newborns (except for the expired) demonstrates the usefulness of newborn screening by MS/MS for early diagnosis and medical intervention. However, close coordination between the MS/MS screening laboratory and the metabolic clinic/biochmical geneticists is needed to determine proper decision of screening parameters, confirmation diagnosis, follow-up scheme and additional tests.

• Journal of Gastric Cancer
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• 제23권3호
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• pp.476-486
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• 2023

Purpose: The optimal tumor mutational burden (TMB) value for predicting treatment response to programmed cell death-1 (PD-1) checkpoint inhibitors in advanced gastric cancer (AGC) remains unclear. We aimed to investigate the optimal TMB cutoff value that could predict the efficacy of PD-1 checkpoint inhibitors in AGC. Materials and Methods: Patients with AGC who received pembrolizumab or nivolumab between October 1, 2020, and July 27, 2021, at Samsung Medical Center in Korea were retrospectively analyzed. The TMB levels were measured using a next-generation sequencing assay. Based on receiver operating characteristic curve analysis, the TMB cutoff value was determined. Results: A total 53 patients were analyzed. The TMB cutoff value for predicting the overall response rate (ORR) to PD-1 checkpoint inhibitors was defined as 13.31 mutations per megabase (mt/Mb) with 56% sensitivity and 95% specificity. Based on this definition, 7 (13.2%) patients were TMB-high (TMB-H). The ORR differed between the TMB-low (TMB-L) and TMB-H (8.7% vs. 71.4%, P=0.001). The progression-free survival and overall survival (OS) for 53 patients were 1.93 (95% confidence interval [CI], 1.600-2.268) and 4.26 months (95% CI, 2.992-5.532). The median OS was longer in the TMB-H (20.8 months; 95% CI, 2.292-39.281) than in the TMB-L (3.31 months; 95% CI, 1.604-5.019; P=0.049). Conclusions: The TMB cutoff value for predicting treatment response in AGC patients who received PD-1 checkpoint inhibitor monotherapy as salvage treatment was 13.31 mt/Mb. When applying the programmed death ligand-1 status to TMB-H, patients who would benefit from PD-1 checkpoint inhibitors can be selected.