• 한국패류학회지
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• 제27권4호
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• pp.387-393
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• 2011

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from $10^5$ in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.

• 한국식품과학회지
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• 제36권5호
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• pp.723-727
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• 2004

콩 가공식품인 두유, 두부, 비지에서 유전자변형 콩에 삽입된 epsps 유전자의 검출과 정량을 위해 정성과 정량 PCR 방법을 수행하였다. 두유, 두부, 비지에서 유전자변형 콩의 검출은 삽입된 epsps 유전자의 증폭이 크기가 121bp와 330bp가 생성될 수 있는 두 종류의 primer들을 이용하여 0.01%까지 확인하였다. 정량방법은 1, 3, 5%의 유전자변형 콩이 포함된 시료들을 실시간 PCR을 사용하여 유의성 있는 결과를 얻었다. 이러한 결과들은 실시간 PCR 방법을 사용하여 가공식품 내에서 정량적으로 유전자변형 콩을 정량하는데 적용될 수 있음을 보였다.

• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.335-341
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• 2007

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.

• 대한임상검사과학회지
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• 제52권4호
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• pp.335-341
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• 2020

본 연구는 Cobas TaqMan MTB 검사(CTM test, Roche Diagnostics, Basel, Switzerland)와 항산균 도말염색검사의 연관성을 확인하고 그에 따른 반정량적 판정기준을 확립하고자 하였다. 2015년 1월부터 2015년 12월까지 삼성서울병원 진단검사의학과에 의뢰된 8,389개의 검체에 대해 결핵균 도말 검사, 배양 검사 및 CTM 검사를 동시에 실시하였으며, 그 결과를 분석하여 AFB 염색과주기의 연관성을 후향적으로 분석하였다. CTM 검사의 임계 값(Ct)값으로, 결핵균 검출방법의 반정량적 판정기준을 설정 하였다. CTM 검사의 135개의 양성 표본에 대한 Ct값은 항산균 도말염색과 반비례적 상관관계가 있었다(rs=-0.545, P<0.01). CTM 검사와 항산균 염색 등급의 음의 상관관계가 입증되었으며, 임상 기준에 이러한 기준을 적용하여 임상적 의의를 검증하였다. 이 연구의 반정량적 기준은 병원에서 활동성 결핵 및 감염을 일으킬 수 있는 환자의 빠른 진단의 판단에 도움이 될 것으로 사료된다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권1호
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• pp.339-342
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• 2012

Background: About one third of the human population suffer cancer during their lifetime and more than 20% of total morbidity is related to neoplasia. Cervical cancer is generally the most common cancer in developing countries and the second most common in women globally. The role of human papilloma viruses viruses in its induction is clear. However, the involvement of hepres simplex virus type 2 (HSV-2) is controversial. Therefore a survey was conducted of the prevalence of HSV-2 in patients with cervical cancer and also healthy people with sensitive and quantitative Taq Man real-time PCR assay. Materials and methods: Seventy six formaldehyde fixed paraffin embedded tissue specimens from patients with histologically proven history of cervical cancer as well as 150 control blocks were sectioned for deparaffinization and DNA extraction. Results: There was no HSV-2 DNA in our patient specimens but four control samples were positive, all with a history of hysterectomy. Conclusion: Considering the absence of any positive viral HSV-2 DNA in our patients and also the presence of four positive specimens among our controls, we did not find any relationship between the presence of HSV-2 DNA and cervical cancer.

• Journal of Veterinary Science
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• 제22권6호
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Journal of Plant Biotechnology
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• 제40권1호
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• pp.18-26
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• 2013

살충성 유전자 mcry1Ac1을 포함하고 있는 해충저항성 유전자변형(GM) 벼 Agb0101이 국내에서 개발되었다. 향후 Agb0101 벼의 환경방출에 따른 모니터링과 이력추적을 위해서는 신뢰성 있는 검출방법의 개발이 필요하다. 따라서, 본 연구에서 해충저항성 GM벼의 사후 안전관리를 위한 정성적 및 정량적 PCR 검정 방법을 개발하였다. 벼 녹말분지효소 유전자 RBE4를 PCR 분석의 내재유전자로 사용하였고, 이의 primer쌍 RBEgh-1/-2는 101bp의 PCR 증폭산물을 형성하였다. 정성 PCR 분석을 위해서 삽입된 T-DNA를 바탕으로 특이 primer를 제작하였고, 이벤트 특이적 검출 primer의 경우 Agb0101의 도입유전자 및 벼 염색체 DNA 사이의 5' 또는 3' 인접염기부위를 정확하게 특이적으로 PCR 증폭하였다. 반면, 대조구인 각종 작물, 국내 벼 품종 및 Agb0101과 동일 형질전환 벡터를 갖는 해충저항성 벼에서는 어떠한 PCR 증폭산물도 형성하지 않았다. 표준물질로써 내재유전자 및 이벤트 특이적 단편으로 제조된 pRBECrR을 이용한 real-time PCR 분석에 의해서 정량한계(LOQ)가 10 copies 농도의 범위인 것으로 확인되었고, 이의 유효성을 검증하기 위하여 상이한 농도의 Agb0101시료(10, 5, 3 및 1%)를 real-time PCR 분석하여 정량검정에 대한 표준편차 및 상대표준편차가 각각 0.06 ~ 0.40 및 3.80 ~ 7.01%의 낮은 범위에 포함되는 것을 확인할 수 있었다. 이들 결과로 본 연구에서 개발된 정성 및 정량 PCR 검정 방법이 해충저항성 GM벼 Agb0101의 모니터링 및 이력추적에 효과적으로 이용될 수 있을 것으로 본다.

• 생명과학회지
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• 제24권8호
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• pp.895-902
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• 2014

아디포넥틴은 이미 합성된 GLUT4의 translocation 증가를 통해 포도당의 세포내 유입을 촉진하며 인슐린 민감도를 증가시키는 것으로 알려져 있다. 본 연구에서는 장기간(6주령부터 16, 26, 36, 47, 및 77주령까지)의 고지방식이(HFD)를 섭취한 비만 C57BL/6 생쥐와, 칼로리제한(CR) 또는 thiazolidinedione (TZD) 섭취에 의해 인슐린 민감성이 회복된 생쥐들로부터 지방조직을 적출하여 아디포넥틴과 GLUT4 의 mRNA 발현의 변화를 조사하였으며, 선형회귀분석(linear regression analysis)을 통해 아디포넥틴과 GLUT4 유전자 발현량 사이의 상관관계를 평가하여 아디포넥틴이 GLUT4 유전자 발현의 전사단계에서도 영향을 미치는지의 가능성을 확인하고자 하였다. 지방조직에서의 유전자 발현량은 TaqMan probe를 이용한 real-time PCR로 정량되었다. 실험결과, 지방조직에서의 아디포넥틴 mRNA발현량은 여러 조건의 생쥐 그룹들 사이에 유의한 변화가 나타나지 않았지만, GLUT4의 유전자 발현량은 HFD군에서는 감소하고, CR군(p<0.05)과 TZD군(p=0.007)에서는 유의하게 증가하는 변화가 확인되었다. 또한, 아디포넥틴과 GLUT4 mRNA 발현량 사이에는 유의한 상관관계를 나타내고 있음이 확인되었다. ND군(p<0.0001), HFD군 p<0.0001), 또는 각각의 주령과 식이별 소그룹, 그리고 CR군(p=0.002) 에서도 두 유전자간의 발현량이 유의하게 연관되어 있었다. 그러나 TZD군(p=0.73)의 생쥐에서는 그 연관성이 사라짐을 관찰하였다. 이는 TZD가 아디포넥틴 유전자 발현에는 영향을 미치지 않지만, GLUT4유전자 발현은 촉진하기에 두 유전자 사이에 유의하지 않은 상관관계로 변화되었음을 시사한다. 이들 결과는 아디포넥틴과 GLUT4의 유전자 발현은 강하게 연관되어 있으며, 두 유전자 발현 조절에 대한 공통적인 작용기전의 존재 가능성 또는 아디포넥틴이 GLUT4 translocation뿐만 아니라 GLUT4의 유전자 발현에도 직접적으로 작용하고 있음을 시사한다.

• 대한두경부종양학회지
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• 제23권1호
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• pp.21-25
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• 2007

Background：Squamous cell carcinoma(SCC)of the palatine tonsils represents approximately 15-23% of all intraoral SCC. The most frequently reported risk factors for oropharyngeal cancer are smoking and alcohol. In a recent overview of HPV and tonsillar squamous cell carcinoma(TC), 51% contained HPV DNA, and HPV-16 being the most frequent type. We aimed to clarify whether HPV directly effects on the oncogenesis and biologic behavior of TC by comparison with infection prevalence, and physical status of virus. Material and Method：We used HPV genotyping DNA chip(Biocore, Korea, Seoul) arrayed by multiple oligonucleotide probes of L1 sequence of 26 types of HPV and HPV genotypes are identified by fluorescence scanner. The copy numbers of HPV E2 and E6 open reading frames(ORF) were assessed using a TaqMan-based 5'-exonuclease quantitative real-time PCR assay. The ratio of E2 to E6 copy numbers was calculated to determine the physical status of HPV-16 viral gene. Results：We observed a significant difference in HPV prevalence between 52 TCs and 69 CFTs(73.1% vs. 11.6%), and most of the HPVs were type 16(87.2%)and non-episomal(94.1%) state. Conclusions：This study regarding HPV infection prevalence and mechanism in the largest population of palatine tonsillar squamous cell carcinoma with chronic follicular tonsillitis revealed significant difference pf HPV prevalence between TC and CFT. Most of HPV were 16 type and integrated or mixed, HPV-16 integration could be directly related to tonsillar carcinogenesis.