• Journal of Korean Society on Water Environment
• /
• v.34 no.1
• /
• pp.46-56
• /
• 2018

A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

• Journal of Life Science
• /
• v.24 no.12
• /
• pp.1269-1275
• /
• 2014

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.3
• /
• pp.595-602
• /
• 2005

A one-step real-time quantitative RT-PCR assay in combination with automated RNA extraction was evaluated for routine testing of HCV RNA in the laboratory. Specific primers and probes were developed to detect 302 bp on 5'-UTR of HCV RNA. The assay was able to quantitate a dynamic linear range of $10^7-10^1$ HCV RNA copies/reaction ($R^2=0.997$). The synthetic HCV RNA standard of $1.84{\pm}0.1\;(mean{\pm}SD)$ copies developed in this study corresponded to 1 international unit (IU) of WHO International Standard for HCV RNA (96/790 I). The detection limit of the assay was 3 RNA copies/reaction (81 IU/ml) in plasma samples. The assay was comparable to the Amplicor HCV Monitor (Monitor) assay with correlation coefficient r=0.985, but was more sensitive than the Monitor assay. The assay could be completed within 3 h from RNA extraction to detection and data analysis for up to 32 samples. It allowed rapid RNA extraction, detection, and quantitation of HCV RNA in plasma samples. The method provided sufficient sensitivity and reproducibility and proved to be fast and labor-saving, so that it was suitable for high throughput HCV RNA test.

• Journal of Life Science
• /
• v.17 no.10
• /
• pp.1374-1380
• /
• 2007

The development of polymerase chain reaction (PCR) technology has the potential to solve for isolating pathogenic microorganisms from environmental samples than traditional plate counting methods. We have been detected pathogenic bacteria from raw water and water treatment process in Busan metropolitan city by PCR method. According to the result of survey from July 2004 to October 2005, 80 out of 92(87.0%) were positive for bacterial pathogens in raw water samples and positive rate of Shigella spp., Yersinia spp., Salmonella spp. and Legionella spp. were 46.2%, 40.7%, 17.6% and 9.9%, respectively. Pathogenic bacteria in raw water was mainly distributed through the lately Autumn to the winter and more highly detected Maeri than Mulgum region. During the period of survey in water treatment process, Shigella spp. was highly detected but all of bacterial pathogens were entirely removed after in post-ozonation step. These suggest that waters supplied in Busan metropolitan city may be safe against the pathogenic bacteria.

• Journal of Veterinary Science
• /
• v.21 no.2
• /
• pp.22.1-22.9
• /
• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.12
• /
• pp.1791-1797
• /
• 2007

A study was conducted with 70 Hanwoo (Korean cattle) for genotyping bovine leukocyte antigen (BoLA)-DRB3.2 gene by using the polymerase chain reaction (PCR) and sequence based typing (SBT). Two-step PCR was carried out for amplifying a 284 bp fragment of the target gene and the PCR products were digested with three restriction enzymes namely RsaI, BstYI and HaeIII. Seventeen alleles were detected with frequencies ranging from 1.43 to 18.57% and one (x'aa) of these alleles was identified as a new allele that has not been reported before. The frequency of the new x'aa allele identified in this breed was 12.86%. In addition, the seven most frequently observed alleles (DRB3.2 *10, *15, *16, *26, *27, *54 and x'aa) accounted for 74.28% of the alleles in this population. The phylogenetic tree showed that the BoLA-DRB3.2 allele sequences of Hanwoo were shared with other Bos taurus breeds and no specific clade for Hanwoo was identified. It indicates high heterogeneity of the BoLA-DRB3 gene in this population and may give some ideas for breeding animals having better disease resistance.

• Korean Journal of Clinical Laboratory Science
• /
• v.52 no.2
• /
• pp.143-149
• /
• 2020

The study examined the seasonal pattern of larvae, nymph, and adult life stages for Haemaphysalis longicornis and the rate of infection with severe fever from the thrombocytopenia syndrome virus in ticks collected from 12 sections (Jichukdong), 14 sections (Uldaeri), and 18 sections (Howondong) in Bukhansan dullegil for April-October 2019. Haemaphysalis longicornis ticks have been considered the main vector for severe fever with thrombocytopenia syndrome (SFTS). Haemaphysalis flava and Ixodes nipponensis were collected using the dragging and flagging method. The ratios of Haemaphysalis longicornis of the collected ticks were 91% (Jichukdong), 94% (Uldaeri), and 98% (Howondong). Monthly distributional studies of Haemaphysalis longicornis based on the developmental stage showed that the adults peaked in September while nymphs were collected more frequently from April through June. The larvae peaked in September and October. SFTS virus detection was performed using 2 × OneStep RT-PCR and nested PCR. On the other hand, no SFTS virus-specific gene was detected in 1,158 ticks of Haemaphysalis longicornis. This result provides estimates of the population densities for the life stages of Haemaphysalis longicornis and the associated disease risk in Bukhansan dullegil, where many people have visited since opening in 2010.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.12
• /
• pp.1953-1957
• /
• 2008

Recombinant Lactobacillus strains have been constructed using gene splicing by overlap extension (SOE). Primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. For efficient matching, we used an asymmetric PCR step that was effective at generating an excess of strands that would anneal in the final PCR. CP12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage A2, was constructed and inserted into the genome of Lactobacillus casei ATCC 393, yielding Lb. casei ATCC 393::XCP12. Another recombinant Lb. casei strain was constructed, where the egfp gene was a part of the construction. The EGFP produced from Lb. casei ATCC 393::XCEGFP14 was detected by Western blot hybridization. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques for Lactobacillus species.

• Molecular & Cellular Toxicology
• /
• v.2 no.4
• /
• pp.251-256
• /
• 2006

The exploration of genes which expressions are changed by exposure to ecotoxicants or pollutants can provide the important information about the reaction mechanisms in the body as well as adaptation to exterior stimulus or environmental changes. Also they can be developed as biomarkers for the detection of environmental pollution. Differential display polymerase chain reaction (DD-PCR) technique has been usefully used to hunt the clones which expressions are up-regulated or down-regulated by exterior changes and this study aimed to search for those clones in diesel oil-exposed rockfish (Sebastes schlegeli) using DD-PCR. The RNA isolated from liver of 20 ppb diesel oil-exposed rockfish was used for screening of the differentially displayed genes and total 44 differentially expressed genes (DEG) are detected then their nucleotide sequences were analyzed. The present data provided the general information about the effect of diesel oil contamination on marine organism and further more the primary step in development of new biomarkers for marine environmental pollution or ecotoxicological stresses.

• Korean Journal of Food Science and Technology
• /
• v.39 no.1
• /
• pp.71-76
• /
• 2007

Foodborne illness caused by Noroviruses (NVs) is increasing rapidly in Korea. This study developed an effective detection protocol for NVs found in contaminated oysters and lettuce through an investigation using the major steps of virus particle separation, concentration and RT-PCR. As a surrogate model for NVs, the cultivable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Instead of using a time-consuming ultracentrifugation method, efficient methods based on solvent extraction and PEG precipitation procedure were applied. Direct homogenization of a 25g sample of whole oyster and lettuce in 175mL PBS provided the simplicity that would be needed in the actual field of food product examination. The overnight PEG precipitation step at $4^{\circ}C$ was reduced to 3 h by placing the reaction tube in ice and by adjusting the PEG concentrations. The application of the use of chloroform and 0.2 ${\mu}m$ syringe filtration together showed a better detection efficiency than the use of chloroform alone in removing PCR inhibitors for both oyster and lettuce samples. Also, dilution of the extracted RNA solution before PCR provided increased sensitivity. The improved detection protocol developed in this study could be efficiently applied to detect FCV and most likely NVs from oysters and lettuce.