• The Plant Pathology Journal
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• v.35 no.1
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• pp.41-50
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• 2019

Sugarcane bacilliform viruses (SCBV), which belong to the genus Badnavirus, family Caulimoviridae, are an important DNA virus complex that infects sugarcane. To explore the genetic diversity of the sugarcane-infecting badnavirus complex in China, we tested 392 sugarcane leaf samples collected from Fujian, Yunnan, and Hainan provinces for the occurrence of SCBV by polymerase chain reaction (PCR) assays using published primers SCBV-F and SCBV-R that target the reverse transcriptase/ribonuclease H (RT/RNase H) regions of the viral genome. A total of 111 PCR-amplified fragments (726 bp) from 63 SCBV-positive samples were cloned and sequenced. A neighbor-joining phylogenetic tree was constructed based on the SCBV sequences from this study and 34 published sequences representing 18 different phylogroups or genotypes (SCBV-A to -R). All SCBV-tested isolates could be classified into 20 SCBV phylogenetic groups from SCBV-A to -T. Of nine SCBV phylogroups reported in this study, two novel phylogroups, SCBV-S and SCBV-T, that share 90.0-93.2% sequence identity and show 0.07-0.11 genetic distance with each other in the RT/RNase H region, are proposed. SCBV-S had 57.6-92.2% sequence identity and 0.09-0.66 genetic distance, while SCBV-T had 58.4-90.0% sequence identity and 0.11-0.63 genetic distance compared with the published SCBV phylogroups. Additionally, two other Badnavirus species, Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV), which originally clustered in phylogenetic groups SCBV-E and SCBV-F, respectively, are first reported in China. Our findings will help to understand the level of genetic heterogeneity present in the complex of Badnavirus species that infect sugarcane.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

• Korean Journal of Plant Resources
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• v.14 no.1
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• pp.65-70
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• 2001

This study was performed to select marker which can identify genetic variation between mother plant and in vitro cultured plantlets of strawberry by PCR using random primer. When 'Yeobong' DNA extracted was treated with proteinase-K and RNase-H, clear DNA bands were shown. The optimal condition for RAPD in strawberry was to use 50ng of template DNA, 10pmol of primer,37oC of annealing temperature, and 45 cycles of PCR. After establishing above PCR optimal condition, RAPD pattern was investigated by using UBC primers. PCR was performed, and 46 of 90 primers produced PCR product showing 158 total bands. GC content was compared between the primers forming bands and no bands. The GC content showing bands was average 67.4%, whereas primers showing no bands 58%.

• Food Science of Animal Resources
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• v.31 no.6
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• pp.851-857
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• 2011

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk ($10^7$ cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as $10^3$ cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli ($10^7$ cells/mL) were heated at $65^{\circ}C$ for 30 min. In addition, subsequent incubation at $37^{\circ}C$ for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at $37^{\circ}C$ for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

• KSBB Journal
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• v.24 no.5
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• pp.432-438
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• 2009

Here, we demonstrated simple nucleic acid, RNA, concentration method using polymer micro chip containing glass bead ($100\;{\mu}m$). Polymer micro chip was fabricated by PDMS ($1.5\;cm\;{\times}\;1.5\;cm$, $100\;{\mu}m$ in the height) including pillar structure ($160\;{\mu}m\;(I)\;{\times}\;80\;{\mu}m\;(w)\;{\times}\;100\;{\mu}m\;(h)$, gap size $50\;{\mu}m$) for blocking micro bead. RNA could be adsorbed on micro glass bead at low pH by hydrogen bonding whereas RNA was released at high pH by electrostatic force between silica surface and RNA. Amount of glass beads and flow rate were optimized in aspects of adsorption and desorption of RNA. Adsorption and desorption rate was measured with real time PCR. This concentrated RNA was applied to amplification micro chip in which NASBA (Nucleic Acid Sequence Based Amplification) was performed. As a result, E.coli O157 : H7 in the concentration of 10 c.f.u./10 mL was successfully detected by these serial processes (concentration and amplification) with polymer micro chips. It implies this simple concentration method using polymer micro chip can be directly applied to ultra sensitive method to measure viable bacteria and virus in clinical samples as well as environmental samples.

• Plant Resources
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• v.4 no.3
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• pp.192-195
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• 2001

Human immunodeficiency virus (HIV), the causative agent of AIDS, still continues to spread rapidly in the world population, especially in Africa and Southeast Asia. At present, two kinds of therapeutic approaches are used for treatment of AIDS. One is to target HIV reverse transcriptase, which is responsible for the viral genome transcription. The other is to inhibit HIV pretense PR, which is essential for the processing of viral proteins. Drug combinations based on these approaches can reduce the blood virus to an undetectable level. However, a small amount of virus may lurk inside the immune cells in a dormant state. Another major obstacle of long-term treatment of the disease is remarkable mutation in HIV. Most of the clinical chemotherapeutic agents have one or more of these problems. High cost and harmful side-effects further reduced the desirability of these drugs. In the course our studies on development of anti-HIV agents from natural products, we investigated various crude drugs for their inhibitory activity against HIV-induced cytopathic effects (CPE) in culture cells, HIV-pretense (PR), HIV-reverse transcriptase (RT) including ribonuclease H (RNase H), and HIV integrase (INT). In the present paper, some inhibitory substances relating to the development of anti-HIV agents are reported.

• The Korean Journal of Mycology
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• v.20 no.3
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• pp.234-239
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• 1992

The experiment was performed to find out the possibilities to detect virus infection in oyster mushrooms, Pleurotus species by analysis of doublestranded ribonucleic acid (ds RNA). Ds RNA segments were extracted from virus infected isolates which grew abnormally. But virus free isolates didn't show any ds RNA segments. The ds RNA was consisted of one large segment of 8100 base pairs (bp) and 4 smaller segments with 2170, 2120, 1980 and 1984 bp. Whereas, cell free virus particles showed only one larger ds RNA segment. The ds RNA was dissolved by RNase A in low salt, 0.1 M SSC and melted at $85^{\circ}C$. It was possible to use the ds RNA analysis for detecting virus infection directly from the host cells.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.68-72
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• 2002

To obtain a yeast mutant with high RNA content and high growth rate, Saccharomyces cerevisiae MTY62 was mutated with ethylmethane sulfonate. Among the selected mutants that were sensitive to the high concentration of KCl, M40-10 strain was finally selected due to its rapid cell growth and high RNA content in the tube and baffled-flask cultures. In the batch culture of M40-10 mutant, the maximum specific growth rate ($\mu_{max}$) of $0.38 h^{-1}$ , RNA concentration of 3210 mg-RNA/1, and RNA content of 183 mg-RNA/g-DCW were obtained, which were 23%, 15%, and 12% increased levels, respectively, compared to those of MTY62 parent strain. The intermittent fed-batch culture of M40-10 strain resulted in the maximum cell concentration of 35.6 g-DCW/1, RNA concentration of 5677 mg/1, and RNA content of 160 mg-RNA/g-DCW. Through the constant fed-batch culture, the maximum cell concentration of 46.4 g-DCW/1, RNA concentration of 6270 mg-RNA/1, and RNA content of 135 mg-RNA/g-DCW were obtained. At the 20 h culture time in the fed-batch cultures of M40-10 strain, the cell and RNA concentrations were increased by 30% and 10%, respectively, over the parent strain MTY62. In addition, it was also found that the accumulated RNA within the mutant cell was not degraded until the end of fed-batch cultivation, indicating that the M40-10 cell is a mutant with weak acidic RNase activity.y.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.878-884
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• 2013

Salmonella is a major foodborne pathogen that causes a variety of human diseases. Development of ligands directly and specifically binding to the Salmonella will be crucial for the rapid detection of, and thus for efficient protection from, the virulent bacteria. In this study, we identified a RNA aptamer-based ligand that can specifically recognize Salmonella Typhimurium through SELEX technology. To this end, we isolated and characterized an RNase-resistant RNA aptamer that bound to the OmpC protein of Salmonella Typhimurium with high specificity and affinity ($K_d$ ~ 20 nM). Of note, the selected aptamer was found to specifically bind to Salmonella Typhimurium, but neither to Gram-positive bacteria (Staphylococcus aureus) nor to other Gram-negative bacteria (Escherichia coli O157:H7). This was evinced by aptamer-immobilized ELISA and aptamer-linked precipitation experiments. This Salmonella species-specific aptamer could be useful as a diagnostic ligand against pathogen-caused foodborne sickness.