• Journal of Microbiology
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• 제44권1호
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• 대한의생명과학회지
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• 제8권3호
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• pp.189-193
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• 2002

We have identified and analyzed the 5'-coding region of SCN5A gene in Korean genome. Although its sequence has already been reported in western countries it is still important to confirm our own sequence for the establishment of Korean-suitable diagnosis on genetic basis. Total RNAs were obtained from three healthy Korean adult hearts and reversely transcribed. RT products were then subjected to PCR reaction followed by DNA sequencing. Three different sets of SCN5A primers were designed and used for the amplification of 5'-coding region of SCN5A from Korean genome. Amplified sequence was roughly one-10th of the entire SCN5A mRNA in size and its detailed sequence was completely matched up to the previously reported sequence. There was no difference between three heart samples, either. So, SCN5A was concluded as the relatively stable gene comparing to other genes that are involved in long QT syndrome.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.900-908
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• 2016

The effects of algal inoculation on chemical oxygen demand (COD) and total nitrogen (TN) removal, and indigenous bacterial dynamics were investigated in municipal wastewater. Experiments were conducted with municipal wastewater inoculated with either Chlorella vulgaris AG10032, Selenastrum gracile UTEX 325, or Scenedesmus quadricauda AG 10308. C. vulgaris and S. gracile as fast growing algae in municipal wastewater, performed high COD and TN removal in contrast to Sc. quadricauda. The indigenous bacterial dynamics revealed by 16S rRNA gene amplification showed different bacterial shifts in response to different algal inoculations. The dominant bacterial genera of either algal case were characterized as heterotrophic nitrifying bacteria. Our results suggest that selection of indigenous bacteria that symbiotically interact with algal species is important for better performance of wastewater treatment.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.381-384
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• 2011

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial largesubunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

• 생약학회지
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• 제47권2호
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• pp.137-142
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• 2016

Enterovirus is a common cause of several severe diseases such as myocarditis, hand-foot-mouth disease, and meningitis in children and adult. There are many try to develop new antiviral drug for direct treatment in virus infection. However, synthetic chemical antiviral drug is not working. To overcome this limitation, we examined plant extracts. The antiviral effect of plant extracts was screened by HeLa cell survival assay in coxsackievirus B3 (CVB3) infection. We observed a strong antiviral effect of Poria cocos extract in a dose-dependent manner (1 mg/ml~0.01 mg/ml). P. cocos extract (1 mg/ml) treatment was dramatically decreased virus protease 2A induced eIF4G-I cleavage and virus capsid protein VP1 production. CVB3 positive and negative strand RNA amplification were significantly reduced in P. cocos extract treatment. P. cocos extract completely blocked early time activation of ERK and AKT activity in CVB3 infection. Taken together these data indicate that the treatment of P. cocos extract strongly inhibit CVB3 replication. Poria cocos extract may possible to developed as a therapeutic agent for enterovirus.

• The Plant Pathology Journal
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• 제19권3호
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• Preventive Nutrition and Food Science
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• 제15권2호
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• pp.159-166
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• 2010

The aim of this study was to develop species-specific primer sets for kimchi Lactobacillus. Known gene sequences of Lactobacillus 16S rRNA were collected from the NCBI Gene bank, and 69 primer sets were designed using the homologous gene sequence. Six species of kimchi Lactobacilli were used as reference strains: Lactobacillus brevis KCTC3102, Lactobacillus farciminis KCTC3681, Lactobacillus fermentum KCTC3112, Lactobacillus hilgardii KCTC3500, Lactobacillus plantarum KCTC3099, and Lactobacillus sanfranciscensis KCTC3205. PCR amplification and gel electrophoresis were performed to identify the accuracy and specificity of the developed primer set. The results show that the primer set of 5'-aagcctgcgaaggcaag-3' & 5'-aggccaccggctttg-3', 5'-acatactatgcaaatctaagagattagacg-3' & 5'-actgagaatggctttaagagattagcttac-3' resulted in a specific PCR band on L. hilgardii, and primer set of 5'-ctaataccgcataacaactactttcacat-3' & 5'-aacttaataaaccgcctacattctctttac-3' on L. farciminis. The results indicate that the developed primer sets can provide a useful tool for the identification and differentiation of L. hilgardii and L. farciminis from other Lactobacillus species of kimchi.

• Fisheries and Aquatic Sciences
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• 제13권4호
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• pp.263-271
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• 2010

G protein-coupled receptors (GPCR) constitute the largest superfamily of cell membrane receptors, mediating diverse signal-transduction pathways. The melanocortin 4 receptor (MC4R) has been of interest for its physiological role and size, one of the smallest among the GPCRs, which makes it a good model system for the structural study of GPCRs. To study the molecular structure and tissue-specific expression of MC4R in olive flounder (Paralichthys olivaceus), the full-length MC4R gene was obtained using PCR amplification of genomic DNA as well as cDNA synthesis. Sequence analysis of the gene indicates that 978 bp of the MC4R gene encodes 325 amino acids without introns. Sequence alignment with the MC4Rs from other fish shows the highest degree of identity (96%) between Paralichthys olivaceous and Verasper moseri, followed by Takifugu rubripes and Tetraodon nigroviridis (89%). RNA was isolated from various tissues to examine the tissue distribution of MC4R by using RT-PCR. The results showed major expression of MC4R in the liver, brain, and eye, which is consistent with the expression pattern in other fish belonging to the order Pleuronectiformes.

• Journal of Applied Biological Chemistry
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• 제49권3호
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• pp.75-81
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• 2006

A full-length cDNA encoding dihydroflavonol 4-reductase (st-dfr) of potato was isolated by rapid amplification of cDNA ends, and their expression was investigated from purple-fleshed potato (Solanum tuberosum L. cv. Jashim). The st-dfr exists as a member of a small gene family and its transcripts was abundant in the order of tuber flesh, stem, leaf, and root. The expressions of st-dfr gene were light inducible and cultivar dependant. Transgenic potato plants harboring antisense st-dfr (AS-DFR) sequences were analyzed. The accumulation of mRNA was nearly completely inhibited as a result of introducing an AS-DFR gene under the control of the 35S CaMV promoter into the red tuber skin Solanum tuberosum L. cv. Desiree. The anthocyanin content of the tuber peels of the transgenic lines was dramatically decreased by up to 70%. The possible production of flavonols in the peels of AS-DFR transgenic potatoes was discussed.

• 센서학회지
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• 제21권5호
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• pp.359-366
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• 2012

Nanopore DNA sequencing is an emerging and promising technique that can potentially realize the goal of a low-cost and high-throughput method for analyzing human genome. Especially, solid-state nanopores have relatively high mechanical stability, simple surface modification, and facile fabrication process without the need for labeling or amplification of PCR (polymerized chain reaction) in DNA sequencing. For these advantages of solid-sate nanopores, the use of solid-state nanopores has been extensively considered for developing a next generation DNA sequencing technology. Solid-state nanopore sequencing technique can determine and count charged molecules such as single-stranded DNA, double-stranded DNA, or RNA when they are driven to pass through a membrane nanopore between two electrolytes of cis-trans chambers with applied bias voltage by measuring the ionic current which varies due to the existence of the charged particles in the nanopore. Recently, many researchers have suggested that nanopore-based sensors can be competitive with other third-generation DNA sequencing technologies, and may be able to rapidly and reliably sequence the human genome for under $1,000.