• Journal of Microbiology and Biotechnology
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• v.31 no.12
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• pp.1716-1721
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• 2021

Chikungunya fever is an arboviral disease caused by the Chikungunya virus (CHIKV). The disease has similar clinical manifestations with other acute febrile illnesses which complicates differential diagnosis in low-resource settings. We aimed to develop a rapid test for CHIKV detection based on the nucleic acid lateral flow immunoassay technology. The system consists of a primer set that recognizes the E1 region of the CHIKV genome and test strips in an enclosed cassette which are used to detect amplicons labeled with FITC/biotin. Amplification of the viral genome was done using open-source PCR, a low-cost open-source thermal cycler. Assay performance was evaluated using a panel of RNA isolated from patients' blood with confirmed CHIKV (n = 8) and dengue virus (n = 20) infection. The open-source PCR-NALFIA platform had a limit of detection of 10 RNA copies/ml. The assay had a sensitivity and specificity of 100% (95% CI: 67.56% - 100%) and 100% (95% CI: 83.89% - 100%), respectively, compared to reference standards of any positive virus culture on C6/36 cell lines and/or qRT-PCR. Further evaluation of its performance using a larger sample size may provide important data to extend its usefulness, especially its utilization in the peripheral healthcare facilities with scarce resources and outbreak situations.

• Research in Plant Disease
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• v.10 no.4
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• pp.231-240
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• 2004

In the year of 2002 annual nationwide survey of virus diseases occurring in the pepper fields and greenhouses in Korea, the distribution and the incidence of viral diseases was investigated. The pepper samples from both greenhouses (155 samples) and open fields (227 samples) were collected and further analyzed to detect eleven different viruses by RT-PCR. The results indicate that no sample collected from both greenhouse and open field seems to be infected by TMV, RMV, PVY, AMV, and TSWV. On the other hand, CMV, BBWV2, PepMoV, PMMoV, TMGMV and ToMV are readily identified from greenhouse and open field samples by RT-PCR. The infection rates of the collected samples between greenhouse and open field are largely different. Comparing with 10% of virus-infected pepper samples grown in greenhouse, approximately one third of pepper samples collected from open field are infected. The mixed-infection rates in the virus-infected greenhouse and open field samples are 16% and 61%, respectively. The dominant virus occuring in greenhouse is PMMoV, indicating that virus-infected seed stocks and infected plant debris in the growing area may be important sources of inocula. On the other hand, both CMV and BBWV2 are dominant viruses in open field. This may indicate that the migration of viruliferous insect vectors into pepper fields may be the most important source of inoculum. Also, the survey shows that BBWV2 is newly immerging virus to be controlled in Korea. The discrepancies on the distribution and the occurrence of viral diseases between field and greenhouse may provide a fact that the accumulation and distribution of inoculum by successive cultivation and the migration of viruliferous vectors into growing areas are likely to be important factors to determine the incidence of viral diseases. Therefore, the further studies on epidemiology and the consideration of new breeding program of pepper are essential to minimize virus diseases.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.235-239
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• 2006

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast to in vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately $650{\mu}g/mL$ of protein was produced after 2h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.

• Korean Journal of Environmental Biology
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• v.22 no.1
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• pp.213-219
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• 2004

The bacterial community of water stream, soil and guano in Gossi cave was examined by using PCR amplified the 16S rDNA-denaturing gradient gel electrophoyesis (DGGE). In this study, the genetic diversity and the similarity of bacterial community between open area and non - open area toy cave tour were investigated, and the seasonable variation pattern was compared each other. DGGE is attractive technique, as it sepayate same length dsDNA according to sequence variation typical 16S rDNA genes. The diversity and similarity of bacterial community in cave was analyzed by GC341f and PRUN518r primer sets foy amplification of V3 region of eubacteria 16S rDNA. The specific DGGE band profile of the cave water gives the possibility that the specific bacterial cell can be adapting to the specific cave environment and living in the cave. The DGGE band profiles of all samples with guano were compared and analyzed by image analyzer, in which mutual band profile was compared to be and the band intensity of guano was the highest. From these result, it is thought that the guano was main nutrient source and influenced on the community structure of the cave environment where is nutritionally limited. Pseudomonas sp. NZ060, Pseudomonas pseudoalcaligenes, uncultured Variovorax sp. and soli bacterium NS7 were identified to be on some sample from analysing DNA sequence of some DGGE band.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1189-1196
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• 2005

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

• Journal of Animal Science and Technology
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• v.47 no.2
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• pp.177-186
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• 2005

Xenotransplantation may help to overcome the critical shortage of human tissues and organs for human transplantation, Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply, However, the use of organs across the species barrier may be associated with the risk of transmission of pathogens, specially porcine endogenous retroviruses (PERVs).• Although most of these potential pathogens could be eliminated by pathogen-free breeding, PERVs are not eliminated by this treatment. PERVs are integrated into the genome of all pigs and produced by normal pig cells and infect human cells. They belong to gamma retroviruses and are of three classes viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from pigs from domestic pigs in Korea. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.l-TOPO vectors and sequenced. A total of 91 env clones were obtained from domestic pigs, Berkshire, Duroc, Landrace and Yorkshire in Korea. Phylogenetic analysis of these genes revealed the presence of only PERV class A and B in the proportion of 58 % and 42 %, respectively. Among these, 28 clones had the correct open reading frame: 18 clones in class A and 10 clones in class B. Since both these PERV classes are polytropic and have the capacity to infect human cells, our data suggest that proviral PERVs have the potential to generate infectious viruses during or after xenotransplantation in human.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.1-7
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• 2005

Methylophaga aminosulfidovorans SK1 (KCTC 10323 BP) can utilize trimethylamine as a sole carbon, nitrogen, and energy source. The bacterial flavin-containing monooxygenase (bFMO) gene was identified in the strain and the recombinant enzyme expressed in E. coli oxidized trimethylamine. To study the function and regulation of the bfmo, over 8,000 nucleotide sequences of the neighboring regions including the bfmo were determined. Three open reading frames proceeding to the bfmo gene encoded analogues to highly conserved nitrate/nitrite sensing two-component system regulators and a methyl accepting protein. Two small open reading frames just downstream of the bfmo gene showed no similar proteins of known functions but the sequences were conserved among other bacteria. Reverse transcription-polymerase chain reaction analysis showed that the six putative genes consisted of three transcription units. The three regulatory genes located upstream of the bfmo gene formed two separate transcription units. The bfmo and the two downstream genes were transcribed from a single promoter.

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.121-126
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• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.