• Journal of fish pathology
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• v.5 no.2
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• pp.143-152
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• 1992

Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.

• Biomedical Science Letters
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• v.13 no.1
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• pp.33-38
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• 2007

The aim of this work was to validate a rapid and an accurate method for detecting Mycobacterium leprae in clinical specimens using nested PCR targeting M. leprae-specific repetitive element (RLEP) sequence. The primers were derived from the RLEP sequence which yield a 272 bp outer product and a 230 bp inner product. The specificity and the sensitivity of the nested PCR were compared with those of single PCR for detecting M. leprae using DNAs isolated from reference strain and various species of Mycobacterium. The results showed that the sensitivity of the nested PCR was about 100 to 1,000 times higher than that of the single PCR and also showed that both the single and the nested PCR were highly specific to M. leprae. Subsequently, the usefulness of the single and nested PCR was evaluated with clinical samples isolated from leprosy patients. The number of positive detections by the single and the nested PCR with a total of 20 specimens from leprosy patients were 9 (45%) and 20 (100%), respectively. The results clearly showed that nested PCR has highest sensitivity in detecting M. leprae from clinical specimens. Therefore, nested primers targeting RLEP sequence developed in this study seems to be useful to detect the presence of M. leprae.

• Journal of Mushroom
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• v.13 no.3
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• pp.175-180
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• 2015

Twenty Inter simple sequence repeat (ISSR) primers were used to assess genetic diversity of 64 Agaricus strains including 45 A. bisporus strains and other 19 Agaricus spp. ISSR primers, (GA)T, (AG)YC, (GA)C and (CTC) amplified PCR polymorphic bands between the Agaricus species or within A. bisporus strains. PCR polymorphic bands were inputted for UPGMA cluster analysis. The varieties, Saea, Saedo, Saejeong and Saeyeon that have recently been developed in Korea were involved in the same group with closely genetic relationship of coefficient similarity over 0.92, whereas, other Korean strains were genetically related to A. bisporus strains that were introduced from USA, Eroupe and Chinese. Furthermore, ISSR-PCR polymorphism could potentially be used to identify homokaryon isolates.

• Journal of Plant Biology
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• v.38 no.3
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• pp.267-274
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• 1995

Based upon the nucleotide sequence of As strain of cucumber mosaic virus (CMV-As0 RNA4, coat protein (CP) gene was selected for the design of oligonucleotide primers of polymerase chain reaction (PCR) for detection and identification of the virus. Reverse transcription and polymerase chain reaction (RT-PCR) was performed with a set of 18-mer CMV CP-specific primers to amplify a 671 bp fragment from crude nucleic acid extracts of virus-infected leaf tissues as well as purified viral RNAs. The minimum concentrations of template viral RNA and crude nucleic acids from infected tobacco tissue required to detect the virus were 1.0 fg and 1:65,536 (w/v), respectively. No PCR product was obtained when potato virus Y-VN RNA or extracts of healthy plants were used as templates in RT-PCR using the same primers. The RT-PCR detected CMV-Y strain as well as CMV-As strain. Restriction analysis of the two individual PCR amplified DNA fragments from CMV-As and CMV-Y strains showed distinct polymorphic patterns. PCR product from CMV-As has a single recognition site for EcoRI and EcoRV, respectively, and the product from CMV-Y has no site for EcoRI or EcoRV but only one site for HindIII. The RT-PCR was able to detect the virus in the tissues of infected pepper, tomato and Chinese cabbage plants.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.111-118
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• 1997

Four separate pairs of oligonucleotide primers within the coding region in a T sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme 3nd Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. morginnle and 3. ouata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive. PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

• Journal of Life Science
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• v.14 no.1
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• pp.110-114
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• 2004

The optimized RAPD-PCR conditions, that can be utilized as a basic information for analysis of the gelletic characteristics were developed for genetic analysis of four mulberry varieties, named Milsung, Chungil, Suil, and Hansung using a primer, OPY15 (5'-AGTCGCCCTT-3') from Operon company. We tested several different factors for best PCR condition including concentrations of DNA, primer, Mgclu annealing temperature, number of PCR cycle, and prosence/absence of pre-heating time at the begining of PCR reaction in the $25 \mul$volume. The best RAPD profiles were obtained using 50 ng of DNA, 1 $\mu$M of primer, $1 \mum$of $MgCl_2\;,45^{\circ}C$ of annealing temperature and an absence of pre-heating time. An establishment of the stable and reproducible RAPD-PCR conditions are expected to be useful for the subsequent RAPD-related investigation, such as genetic characterization of the mulberry varieties, re-establishment of phylogenetic relationships and development of new varieties.

• Analytical Science and Technology
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• v.12 no.3
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• pp.260-264
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• 1999

A simple and rapid procedure, called FoLT-PCR(Formamide Low Temperature-Polymerase Chain Reaction) was applied to amplifying DNA directly from various forensic biological evidences including human blood, saliva, hair root, or semen without any DNA preparative steps. We added washing step with non-ionic detergent, 1% Triton X-100, and used Taq DNA polymerase instead of Tth DNA polymerase to amplify 3 STR loci and gender allele simultaneouly. Optimal concentration of formamide and annealing temperature were determined empirically to 8%(v/v), and $48^{\circ}C$ respectively. We also compared this method with standard PCR.

• Journal of the Korean Chemical Society
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• v.47 no.2
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• pp.127-132
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• 2003

We have developed a direct polymerase chain reaction (PCR) and electrophoresis method for the diagnosis of bovine theileriosis from whole blood DNA analysis without DNA extraction. The technique empolyed a FoLT (formamide, low temp.) technique and was utilized in the diagnosis of bovine theileriosis. Formamide solubilize the blood cells, and the lowered incubation temperatures reduced protein coagulation. 100-200 nL of whole blood and PCR reagents were introduced directly into a PCR tube. After the amplification, the PCR product (816-bp DNA) was introduced into the electrophoresis system. The results of this analysis were consist with those obtained using purified DNA.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• The Korean Journal of Mycology
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• v.46 no.2
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• pp.186-192
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• 2018

Rapid and accurate detection methods for Colletotrichum coccodes, an anthracnose pathogen of pepper and tomato, were developed using PCR. A specific primer set, coccoTef-F/coccoTef-R, which was constructed by analyzing tef-$1{\alpha}$ genes from 13 species and 22 strains of Colletotrichum, could specifically detect C. coccodes at a level of 10 ng by conventional PCR method and at 10 pg by real-time PCR. The PCR-based methods were also capable of detecting C. coccodes in pepper and tomato seeds artificially infected with the pathogen. The developed PCR methods can be applied for rapid and accurate inspection of C. coccodes in the seeds intended for export or import.