• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.610-620
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• 2021

There has been increasing interest in the head and neck squamous cell carcinoma (HNSCC) that is caused by high-risk human papillomavirus (HR-HPV) and has posed a significant challenge to Otolaryngologists. A rapid, sensitive, and reliable method is required for the detection of HR-HPV in clinical specimens to prevent and treat HPV-induced diseases. In this study, a multiple cross-linking spiral amplification (MCLSA) assay was developed for the visual detection of HPV-16. In the MCLSA assay, samples were incubated under optimized conditions at 62℃ for 45 min, and after mixing with the SYBR Green I (SGI) dye, the positive amplicons showed bright green fluorescence while the negative amplicons exhibited no obvious change. The specificity test revealed that the developed MCLSA technique had high specificity and could effectively distinguish all five HPV-16 strains from other pathogenic microorganisms. In terms of analytical sensitivity, the limit of detection (LoD) of MCLSA assay was approximately 5.4 × 101 copies/tube, which was 10-fold more sensitive than loop-mediated isothermal amplification (LAMP) and RT-PCR. The detection results of laryngeal cancer specimens collected from 46 patients with suspected HPV infection in the Liaoning region demonstrated that the positive detection rates of MCLSA and hybridized capture 2 kit were 32.61% (15/46). The true positive rate of the MCLSA assay was higher than that of RT-PCR (100% vs. 93.33%) and LAMP (100% vs. 86.67%). Therefore, the MCLSA assay developed in the present study could be a potentially useful tool for the point-of-care (PoC) diagnosis of HR-HPV, especially in resource-limited countries.

• 미생물학회지
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• 제41권3호
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• pp.216-224
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• 2005

혈장분획제제 중 혈액응공인자제제와 일부 면역글로불린제제는 혈장에 존재하는 다양한 단백질로부터 유효한 단백성분만을 선택적으로 분리 정제하기 위해 크로마토그래피 방법을 사용하여 생산된다. 효율적인 세척(cleaning) 공정이 이루어지지 않는다면 크로마토그래피는 다양한 종류의 불순물뿐만 아니라 혈액 중 내재 또는 오염 가능성이 있는 위해인자가 오염될 가능성이 있다. 본 연구에서는 혈장분획제제 제조공정에 사용되는 크로마토그래피의 세척 공정에서 혈장유래 바이러스의 제거 및 불활화 공정의 검토 강화로 혈장분획제제의 안전성을 확보하기 위해 크로마토그래피 세척 검증 시스템을 구축하고자 하였다. 크로마토그래피 세척 공정 중 바이러스 제거 검증을 위해 혈장유래 바이러스 중 물리${\cdot}$화학적 처리에 가장 큰 저항성을 갖는 human parvovirus B19의 모델 바이러스의 porcine parvovirus(PPV)를 대상으로 real-time PCR 정량법을 확립하였다. PPV에 특이적인 primer를 선별하였으며 형광염료 SYBR Green I을 사용하여 PPV DNA를 정량하였다. 세포배양법에 의한 감염 역가와 비교한 결과 PCR 민감도는 1.5 $TCID_{50}/ml$이었다. 확립된 검증법의 신뢰성(reliability)을 보증하기 위해 실험법의 특이성(specificity), 재현성(reproducibility) 등을 검증하였다. 구축된 검증시스템을 thrombin 분리${\cdot}$정제를 위한 SP-Sepharose 양이온 크로마토그래피 공정과 factor VIII 분리${\cdot}$정제를 위한 Q-Sepharose 음이온 크로마토그래피 공정에 적용하여 크로마토그래피 세척 검증을 실시하고, 세척 검증 시스템의 적합성을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.339-345
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• 2009

We evaluated the activity and abundance of the crude-oil-degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon(TPH) degradation rate constants(k) of the soils treated with and without H17-1 were $0.103\;d^{-1}$ and $0.028\;d^{-1}$ respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA(16S rRNA), alkane monooxygenase(alkB4), and catechol 2,3-dioxygenase(23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil($\alpha$=0.05,p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.

• 한국식품위생안전성학회지
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• 제23권2호
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• pp.121-128
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• 2008

본 연구는 Lightcycler (Roche)를 이용한 Real-Time PCR(LC-PCR)기법을 통하여 원유시료에서 신속, 정확하게 황색포도상구균을 검출하는 기법을 개발하고자 하였다. coagulase 전구체를 coding하는 113 bp의 coa 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 황색포도상구균 특유의 유전자 검출하는 기법을 개발하였다. 또한 분리된 균주중 메치실린에 내성을 나타내는 균주를 검출하고자 penicillin-binding protein, PBP2a (mecA)를 coding 하는 209 bp의 mecA 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 메치실린내성 황색 포도상구균을 real-time PCR 기법으로 검출하는 기술을 개발하였다. 본 실험에 따르면 647개의 원유시료중 6개의 시료에서 황색포도상구균이 검출되었으며 이중 2개의 시료에서 분리된 황생포도상구균이 메치실린내성 황색포도상구균임을 확인하였다. 또한 DNA 검출한계는 10 fg으로 기존 PCR에 비해 매우 감도가 우수한 것을 확인하였다. 또한 3개의 원유시료에서 돼지나 소의 삼출성 피부염의 원인균인 Staphylococcus chromogenes가 분리되었다.

• Molecular & Cellular Toxicology
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• 제3권4호
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• pp.320-325
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• 2007

Promoter hypermethylation of the $p16^{INK4a}$ gene was investigated in 52 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with colorectal cancer, using the proposed modified the Real-time PCR/SYBR Green detection method presented in this study. In normal tissue, 29 of 52 patients (56%) were methylated and in tumor tissue, 23 of 52 patients (44%) were methylated. The 34 cases (65.4%) showed a concordant DNA methylation pattern in both normal tissue and tumor tissue. Analyzing the association between the clinicopathologic features and DNA methylation status of the $p16^{INK4a}$ gene, the DNA methylation status according to by Duke's stage was different while other clinicopathological characteristics, including the age, sex, tumor stage, and histologic type of the patient were not found to be correlated with $p16^{INK4a}$ methylation. With multivariate logistic regression, it was observed that the DNA methylation status of $p16^{INK4a}$ gene in normal tissue was correlated with the DNA methylation status of the $p16^{INK4a}$ gene in tumor tissue (P=0.026). According to a Kaplan-Meier survival analysis, a difference in the survival rate by DNA methylation status was found, but it was not significant.

• The Plant Pathology Journal
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• 제33권2호
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• pp.184-192
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• 2017

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at $63^{\circ}C$ for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was $10^{-2}J2/0.5g$ of soil, which was 10 times more sensitive than conventional PCR ($10^{-1}J2/0.5g$ of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.164-169
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• 2008

Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.

• 미생물학회지
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• 제47권2호
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• pp.103-109
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• 2011

Pectobacterium carotovorum subsp. carotovorum (PCC)는 세균성 무름병균으로 주로 감자, 양배추 등의 식물에서 질병을 일으킨다. 본 연구에서는 현장에서 신속하게 진단하기 위해 loop-mediated isothermal amplification법을 이용하여 1시간 내에 등온에서 검출 가능한 진단법을 개발하였으며, 이를 PCC-LAMP법이라 명명하였다. PCC의 lytic murein transglycolase 유전자를 특이적으로 증폭시키는 4개의 프라이머를 제작하였으며 최적 온도가 $61^{\circ}C$임을 확인하고 최적 조건을 확립하였다. 최적 조건을 바탕으로 4개의 프라이머가 $1{\times}10^3$ copies까지 검출하는 민감성을 확인할 수 있었다. 본 연구에서 개발된 PCC-LAMP법은 특이성 검사를 통해 PCC만이 특이적으로 검출됨을 확인하였으며, 이는 실제 시료에서도 적용 가능함을 확인하였다. PCC-LAMP법을 통하여 PCC를 신속하고 정확하게 검출함으로써 현장에서 유용하게 적용될 수 있을 것으로 사료된다.

• 한국식품위생안전성학회지
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• 제30권1호
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• pp.103-108
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• 2015

본 연구에서는 김치로부터 W. cibaria 0D17와 W. confusa 0D23를 생화학적 특성 분석과 16s rRNA 염기서열 분석을 통해 분리, 동정하였으며, W. cibaria 0D17와 W. confusa 0D23 배양 상등액이 L. monocytogenes에 대한 항균 효과가 있는 것으로 나타났다. 또한, 실시간 정량 PCR을 통해 W. cibaria 0D17 및 W. confusa 0D23가 L. monocytogenes를 공동 배양했을 때의 생육억제 효과를 분석한 결과, $37^{\circ}C$에서는 생육 억제 효과가 있는 것으로 확인되었다. 따라서, 김치 유래 Weissella spp.가 갖는 L. monocytogenes에 대한 항균 활성에 대한 기초 자료로 활용가능 할 것으로 판단되며, W. cibaria 0D17 및 W. confusa 0D23가 생산하는 bacteriocin 등의 항균 물질 특성에 대한 연구가 추가적으로 진행될 필요가 있을 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권8호
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• pp.987-992
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• 2010

This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.