• Archives of Pharmacal Research
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• v.27 no.7
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• pp.776-780
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• 2004

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFN$\{alpha}$-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the HindIII and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

• Korean Journal of Veterinary Research
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• v.60 no.3
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• pp.109-116
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• 2020

This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the proline-rich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.

• Plant Resources
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• v.4 no.1
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• pp.41-47
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• 2001

This study was conducted to investigate the occurrence of Soil borne wheat mosaic virus(SbWMV) in barley fields in Korea and to examine the host pathogenicity of SbWMV. By using the ELISA test, SbWMV was detected in the six regions : Suwon, Milyang, Jinju, Youngkwang, Iksan, and Chonju. SbWMV was isolated from the two strains, Albori strain from Jinju and Eunpamil strain from Milyang. SbWMV was collected from leaves showing mosaic, yellowing and necrosis stripes. SbWMV was inoculated mechanically on 1∼1.5 leaf stages with leaf-rubbing to identify the host pathogenicity of 36 Korean barley cultivars, a wheat cultivar, two rye cultivars, three Japanese barley cultivars and Chenopodium amaranticola. Viral sympoms of inoculated leaves appeared on moulted loaves about 4 to 6 weeks of inoculation. Baegdong and Tapgolbori, infected from Albori strain and Eunpamil strain infected from Samdobori showed much higher susceptibility than C. amaranticola and C. quinoa which showed ring spots and chlorotic spots respectively. Virus particles were observed by the electron microscope. They were rod-shapes, which are bipartite, of 142 nm or 281 nm in length with 20 nm diameter on infected leaves. Specific detection and identification of SbWMV was set up using the RT-PCR. PCR fragments of SbWMV(0.5kb) were obtained by using the designed primers for SbWMV RNA 2.

• Journal of Life Science
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• v.30 no.1
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• pp.1-9
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• 2020

Pseudomonas syringae pv. actinidiae, the causal agent of a bacterial canker disease in kiwifruit, is subdivided into five genetically distinct populations, namely biovars 1, 2, 3, 5, and 6. Of these, strains belonging to biovar 3 are responsible for a pandemic bacterial canker of kiwifruits since 2008. This study aimed to characterize the structure of the biovar 3 population and investigate the origin of biovar 3 strains isolated in Korea. The genetic variability of fifteen biovar 3 strains, thirteen Korean and two Chinese, were evaluated through random amplified polymorphic DNA (RAPD)-PCR. The RAPD results revealed the presence of eight lineages, designated as subgroups I-VIII, across the biovar 3 strains used in this study. As the strains in subgroups II and III from China were not found in the Korean examples, we concluded that six genetically different biovar 3 subgroups (I, IV, V, VI, VII, and VIII) are present in Korea. In PCR analysis using primers specific to the strains of New Zealand and Europe, Korean strains in subgroups V and VI amplified the relevant DNA bands, suggesting that these were introduced from these two origins, respectively. PCR primers specific to subgroup VIII were developed to monitor the spread of the first biovar 3 strain in Korea, and investigations revealed that this strain was not found in Korea after its first occurrence.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.53-63
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• 1998

Total of 1085 swine sera (1996-1997) from nation-wide were tested for the presence of antibodies to influenza A virus. Fifty nine percent of the tested sera showed seropositive by HI test. Positive sera consisted of 24--- of H3, 15--- of H1, and 20--- of the sample had both antibodies, respectively. Sera collected from various region represented 7~27--- seropositivity to H1N1, 15~25--- to H3N2, respectively. Swine influenza field isolate from nasal swab was characterized antigenically and genetically to elucidate its relatedness with other known strains of influenza A virus. The study was focused on the HA gene which is related to pathogenecity and antigenic variability of the influenza virus. By RT-PCR using influenza A/H1N1 specific primers, influenza virus H1N1 specific DNA fragment was amplified from A/Swine/Iowa/15/30(H1N1), US field isolate but not in H3N2 strain. PCR products were sequenced by dideoxy chain termination method to determine nucleotide homology with other strains of influenza A virus. The US field isolate and A/Swine/Indiana/1726/88 strain had 97--- of nucleotide homology and 98--- of amino acid homology. Based on the results obtained from this experiment, the field isolate was genetically related to A/Swine/Indiana/1726/88 and had higher homology with A/Swine/Indiana/1726/88 than with classical swine influenza virus, A/Swine/Iowa/15/30. The field isolate had no amino acid changes at the antigenic site compare to that of the A/Swine/Indiana/1726/88. The proteolytic enzyme cleavage site between HA1 and HA2 had no alteration and the amino acid arginine was intact. There is no evidence has been found that the field isolate has genetic shift or genetic drift which might altered antigenic determinant.

• Korean Journal of Plant Tissue Culture
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• v.27 no.1
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• pp.7-12
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• 2000

Explants of lettuce (Lactuca sativa L.) were co-cultivated with Agrobacterium tumifacience GV 3101 strain containing nptII gene and cold regulated gene (BN115) from Brassica napus for transformation. Multiple shoots were obtained from the explants in the selection medium (MS basal medium supplemented with 100 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5 mg/L kinetin) after 3 to 4 weeks of co-culture. The putative transgenic shoots were transferred to rooting medium (1/2 MS basal medium supplemented with 100 mg/L kanamycin and 250 mg/L carbenicillin). The selected shoots were tested with PCR analysis using nptll, BN115 primers whether cold-regulated gene was introduced to genome of the plants. The vir G primers were particularly used to check contamination of Agrobacterium during PCR analysis. The nptII and BN115 primers produced the specific PCR bands in the putative transgenic lines but the vir G primers did not. These results confirmed that the PCR products were not the result of contamination with Agrobacterium. Additionally the Southern analysis of the PCR products and RT-PCR analysis proved that the cold-regulated gene was successfully integrated and transcribed in the putative transgenic lettuce plants.

• International Journal of Industrial Entomology and Biomaterials
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• v.11 no.1
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• pp.57-61
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• 2005

A strain of Bacillus thuringiensis that showed signifi­cantly high toxicity to Plutella xylostella was isolated from a dust sample collected from Chinese tobacco warehouse and characterized. The isolate named B. thuringiensis LY-99 was determined to belong to subsp. alesti (H3a3c) by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid and crystal protein patterns of the LY-99 were different from those of the reference strain, subsp. alesti. PCR analysis with specific primers revealed that this isolate contained abundant cry genes including crylAa, crylAc, crylB, crylD, crylE, crylF and cry2 genes, which was absolutely different from cry gene profile of the subsp. alesti. In addition, insecticidal activity of the LY-99 against P. xylostella larvae was about 44 times higher than that of the subsp. alesti.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.804-810
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• 1999

There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. BIB, a Gram-positive PCB degrader. The strains BIB and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B lB, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.423-427
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• 2012

Pseudomonas syringae pv. actinidiae strains, the causal agents of bacterial canker on kiwifruit, were isolated from Korea and Italy in 2011. Among 87 isolates, a total of six representative strains, three from Korea and three from Italy, were identified on the basis of biochemical and physiological tests. Identities were confirmed by PCR using P. syringae pv. actinidiae-specific primers PsaF1/R2, which amplified a 280-bp DNA fragment. The strains isolated from Korea in this study displayed BOX-PCR patterns similar to those isolated from Italy but different from those isolated previously in Korea or the pathotype P. syringae pv. actinidiae strain. The effector hopA1 and hopH1 genes, which are known to be present in strains isolated recently from France and Italy, were also present in P. syringae pv. actinidiae strains, SYS1, SYS2 and SYS4, isolated from Korea in this work. However, no amplicons of the expected size were obtained from strains previously isolated from Korea and Japan. In addition, the Korean strains isolated in this work belonged to haplotype I for the cts gene identical to those strains isolated from recent outbreaks in Italy. These results suggest that P. syringae pv. actinidiae strains isolated from Korea and examined in this work are a new type of strain similar to those found from recent outbreaks in Italy. This is the first report on the occurrence of cts haplotype I strains of P. syringae pv. actinidiae affecting kiwifruit plants in Korea.