• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1782-1786
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• 2015

A new improved PCR method has been developed for the rapid, reliable, and sensitive detection of Venturia nashicola, a destructive pathogen of scab disease in Japanese pear. The translation elongation factor-1 alpha gene-derived PCR primers specifically amplified a 257-bp-sized DNA band of the target gene from the genomic DNA of V. nashicola. No amplicon was produced from the genomic DNA of other Venturia spp. and reference fungal species tested. With the high detection limit of 10 fg DNA content, our real-time method could be used for the quarantine inspection and field monitoring of V. nashicola.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3065-3069
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• 2016

Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP).

• Fisheries and Aquatic Sciences
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• 제17권3호
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• pp.339-344
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• 2014

PCR amplification with universal primer is a useful tool for speciation of symbionts in marine eukaryote coupled with robust separation method such as denaturing high performance chromatography (DHPLC). To overcome the biased amplification, clamping PCR is recommended to suppress the amplification of host gene. In this study, we evaluated the efficiency of rare gene detection for two kinds of clamping probes which were successfully utilized for eukaryotic symbiont analysis: C3 linked nucleotide (C3) and peptide nucleic acid (PNA). PNA was 3-4 orders of magnitude higher than that of C3 tested in clamping efficiency and rare gene detection. This represented that PNA could be a more competent clamping probe for the enhancement of PCR amplification for rare symbiont genes.

• 대한수의학회지
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• 제38권3호
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• pp.565-573
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• 1998

To study the specific tools for the diagnosis of Newcastle disease virus (NDV) in chicken, polymerase chain reaction (PCR) and its presumable conditions were evaluated for the detection of hemagglutinin-neuraminidase (HN) gene of NDV RNA. For these purposes, Kyojeongwon strain of the NDV was propagated in allantoic cavity of SPF embryonating chicken eggs, and viral RNA was extracted from fractionated virus after the allantoic fluids were ultracentrifuged with sucrose gradient. The first-strand cDNA was then made for the HN gene of NDV RNA by reverse transcription at $42^{\circ}C$ for 1 hour using specific primer complementary to the HN gene. The single-stranded cDNA was used as template in the PCR of the HN-DNA, and various conditions of the PCR were evaluated to set up method for the specific detection of the HN-DNA. The PCR conditions promising for the detection of HN gene consist of preheating at $94^{\circ}C$, 5 min, 30 cycles of denaturation at $94^{\circ}C$, 1 min, annealing at $55^{\circ}C$, 1 min and polymerization at $72^{\circ}C$, 2 min, and a cycle of extension at $72^{\circ}C$, 5 min. when NDVs of allantoic fluids without fractionation were applied to the above PCR condition, the HN genes were detected effectively not only from Kyojeongwon but from other velogenic strains such as Herts and a field isolate.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1353-1358
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• 2005

A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of $4.1{\times}10^{3}$ copies/ml of BPW and $3.3{\times}10^{3}$ copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.

• 한국지능시스템학회:학술대회논문집
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• 한국퍼지및지능시스템학회 2004년도 춘계학술대회 학술발표 논문집 제14권 제1호
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• pp.97-100
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• 2004

The identification of the DNA structure as a double-stranded helix consting of two nucleotide chain molecules was a milestone in modern molecular biology. The DNA chip technology is based on reverse hybridization that follows the principle of complementary binding of double-stranded DNA. DNA chip can be described as the deposition of defined nucleic acid sequences, probes, on a solid substrate to form a regular array of elements that are available for hybridization to complementary nucleic acids, targets. DNA chips based on cDNA clons, oligonucleotides and genomic clons have been developed for gene expression studies, genetic variation analysis and genomic changes associated with disease including cancers and genetic diseases. DNA chips for gene expression profiling can be used for functional analysis in human eel Is and animal models, disease-related gene studies, assessment of gene therapy, assessment of genetically modified food, and research for drug discovery. DNA chips for genetic variation detection can be used for the detection of mutations or chromosomal abnormalities in cnacers, drug resistances in cancer cells or pathogenic microbes, histocompatibility analysis for transplantation, individual identification for forensic medicine, and detection and discrimination of pathogenic microbes. The DNA chip will be generalized as a useful tool in clinical diagnostics in near future. Lab-on-a chip and informatics will facilitate the development of a variety of DNA chips for diagnostic purpose.

• Bulletin of the Korean Chemical Society
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• 제33권1호
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• pp.69-75
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• 2012

Five-channel electrochemical chips were fabricated based on the Micro-electromechanical System (MEMS) technology and were used as platforms to develop DNA arrays. Different kinds of thiolated DNA strands, whose sequences were related to white spot syndrome virus (WSSV) gene, were separately immobilized onto different working electrodes to fabricate a combinatorial biosensor system. As a result, different kinds of target DNA could be analyzed on one chip via a simultaneous recognition process using potassium ferricyanide as an indicator. To perform quantitative target DNA detection, a limit of 70 nM (S/N=3) was found in the presence of 600 nM coexisting noncomplementary ssDNA. The real samples of loop-mediated isothermal amplification (LAMP) products were detected by the proposed method with satisfactory result, suggesting that the multichannel chips had the potential for a high effective microdevice to recognize specific gene sequence for pointof-care applications.

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Applied Biological Chemistry
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• 제49권3호
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• pp.192-195
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• 2006

국내에서 개발된 비타민 E 강화 유전자변형 들깨의 정성 PCR 분석법의 개발을 위해 들깨의 내재 유전자로써 KAS-I (Beta-ketoacyl-ACP synthase I)를 선별하였고, 이러한 내재유전자를 특이적으로 증폭시킬 수 있는Primer(Pfru3-F/R)쌍을 이용한 PCR에서 95 bp의 PCR증폭 산물을 얻었으며, 들깨를 포함한 16개 작물에 대해 PCR을 수행한 결과에서 들깨만이 특이적으로 증폭되는 것을 확인하였다. 또한, 비타민 E 강화 유전자변형 들깨에 삽입된 TMT(${\gamma}$-tocopherol methyltransferase) 유전자와 OCS(Octopine synthase) terminator 연결 부위를 증폭시켜 148 bp의 PCR 산물을 얻을 수 있는 primer(TMTO-F/R)를 제작하였으며, 이러한 두 쌍의 primer를 이용하여 국내 개발된 비타민 E 강화 유전자변형 들깨의 PCR 정성 분석법을 확립하였다.

• Journal of Veterinary Science
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• 제21권2호
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• pp.24.1-24.11
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• 2020

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.