• Korean Journal of Environmental Biology
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• v.22 no.2
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• Journal of Plant Biotechnology
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• v.42 no.2
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• pp.117-122
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• 2015

Canola is a crop globally used for production of oil and biofuel. Cultivation area and import volume of living modified (LM) canola have been increasing every year. As canola import dependence has reached 100% in Korea, efforts have been made for safety management of LM canola and ecological risk assessment. We developed a set of multiplex PCR method for simultaneous detection of 5 LM canola events (Topas 19/2, Rf3, Ms8, RT73 and T45) approved in Korea. The multiplex PCR assay developed allows amplification of estimated products of 5 LM canolas from event specific primer sets. Primer extension time was skipped for a time-consuming process and two annealing steps (20 cycles at $55^{\circ}C$ and 20 cycles at $60^{\circ}C$) were performed for yielding the best result which was sufficient to distinguish five LM canolas. Our results suggest that multiplex PCR method provides a cost and time-effective approach for LM canola detection.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.273-278
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• 2000

The aacCl, aacC2, aacC3, and aacC4 genes, which encode aminoglycoside acetyltransferase AAC(3)-I, AA(3)-II, AAC(3)-III, and AAC(3)-IV, respectively, and aerolysin genes in water samples from Juam lake were surveyed by polymerase chain reaction. Surface water was collected from January 1996 to December 1998, and then bacterial DNA was extracted from the water. Twelve samples were tested by PCR to servey the genes for aminoglycoside acetyltransferase and aerolysin in the lake water. The aacC2 gene was detected in 9 of 12 DNA samples. Among 9 samples showing aacC2 positive, 7 samples were associated with Tn3 sequence. However, none of the twelve samples amplified the expected DNA fragment for aacC1, aacC3, and aacC4 genes. PCR primer to detect the aerolysin gene was designed using the conserved region of the genes for aerolysin and hemolysin of Aeromonas spp. This primer set successfully amplified the expected 414 bp PCR product with the DNA samples from the lake water. The aerolysin gene was detected in 7 of 12 DNA samples. When Southern hybridization of the gel with probe was performed, the aerolysin gene was detected in 10 of 12 DNA samples. However, the seasonal fluctuation of these genes was not found.

• Korean Journal of Food Science and Technology
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• v.43 no.1
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• pp.119-123
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• 2011

In this study, performances of culture methods using two selective media and real-time PCR were evaluated for detection of Campylobacter jejuni (C. jejuni) in various food samples. Sausage, ground beef, and radish sprouts inoculated with C. jejuni were enriched in Hunt broth and then streaked onto modified cefoperazone charcoal deoxycholate agar and Preston agar, followed by incubation under microaerobic conditions. The enriched Hunt broth (1 mL) was used in real-time PCR assay. No statistical differences were observed in sensitivity among the two selective media and real-time PCR for sausage and ground beef. However, the number of positives by real-time PCR in radish sprouts was much higher than the two selective media (p<0.05). It appears that real-time PCR could be used as an effective screening tool to detect C. jejuni, particularly in foods with a high number of background microflora such as fresh vegetables.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.291-294
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• 2006

Clostridium perfringens strains were isolated from swine diarrhea in Korea. Three out of nineteen (15.8%) isolates of C. perfringens were found to be enterotoxigenic by PCR analysis. PCR-based genotyping of the three enterotoxigenic isolates of C. perfringens revealed that they were types A, C and D, respectively. These results suggest that various types of enterotoxigenic C. perfringens can cause swine diarrhea, and that the presence of enterotoxigenic type A strain, known to be strongly associated with food poisoning, may cause public health problem in Korea.

• Restorative Dentistry and Endodontics
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• v.24 no.4
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• pp.535-545
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• 1999

Crease of Streptococcus salivarius is believed to play a critical role in bacterial ecology and pH homeostasis in the mouth, and consequently affect the pathogenesis of dental caries and periodontal diseases. Expression of the urease gene is greatly enhanced by low p. f. excess of Carbohydrate, and faster growth. It was observed that urease activity of the strains of S. salivarius that exhibited no of low urease activity was not increased even in low pH condition. In this study, it was hypothesized that the urease gene of the strains is absent, defected, or greatly changed by genetic combination. In order to prove this hypothesis, chromosomes were obtained from 28 S. salivarius strains which had been isolated from normal teeth and carious lesions, subjected to polymerase chain reaction (PCR) using primers encoding highly conserved sequence from ureC, and then the obtained PCR products were compared. The results were as follows: 1. After PCR the strains generated either one of 0.54- and 1.3-kbp PCR products, or none. 2. All 16 strains having a higher urease activity(<50${\mu}mol/min/mg$) produced 0.54-kbp PCR products. 3. Twelve strains without urease activity and with a lower urease activity(<50${\mu}mol/min/mg$) yield either one of 0.54 and 1.3-kbp PCR products, or none. 4. The DNA sequence of the 0.54-kbp PCR product (pCAP-0.54) exhibited 95% identity to the ureC of S. salivarus 57.I; 30bp were found to be different, which led to difference of only 2 amino acids in the sequence. 5. The DNA sequence of the 1.3-kbp PCR product(pCAP-1.3) was found to be highly homologous to the aminopeptidase C gene of Streptococcus thermophilus. Overall results indicate that there are considerable variations of the urease genes from S. salivarus strains and the variations may affect the uncolytic activity of the bacteria directly of indirectly.

• Korean journal of applied entomology
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• v.51 no.4
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• pp.365-370
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• 2012

The two closely related major leguminous crop pests in Korea, Matsumuraeses phaseoli and M. falcana (Lepidoptera: Tortricidae) have very similar morphological characters, which occasionally give rise to a failure in distinguishing between the two. In this study, we report an easy PCR-SSP method to distinguish between them, with a sequence specific primer set (P-SF2, F-SF3, and C-SR3) based on single nucleotide mismatch in 3' terminal base of a primer, which is found in the mitochondrial cytochrome c oxidase subunit I DNA (mtCOI). Through application of this method, each species may be clearly identified in terms of its PCR band size and pattern, only one band (245 bp) for M. falcana and one (409 bp) or two bands (409 bp & 245 bp) for M. phaseoli.

• Korean Journal of Veterinary Research
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• v.49 no.2
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• pp.105-111
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• 2009

In this study, occurrence of canine brucellosis was surveyed in kennels, indoor dogs and stray dogs in Korea, and infrequent restriction site-polymerase chain reaction (IRS-PCR) was applied to analyze DNA polymorphism of Brucella canis (B. canis) isolates. Among a total of 501 dogs tested, B. canis antibodies by both rapid screening agglutination with 2-mercaptoethanol (2-ME RSAT) and immunochromatographic assay were detected in only 14.1% of kennel dogs. There were no seropositive cases in indoor dogs and stray dogs. DNA polymorphism was observed in 16 B. canis isolates by the IRS-PCR. Sixteen isolates were tested with primers, PsalA, PsalC, PsalG and PsalT, and different primers produced different DNA patterns. In regard to the IRS-PCR pattern of 16 isolates, 9 (56.3%) belonged to the IRS-PCR type I. The remaining 7 were differentiated as type II, III and IV. An application of the primer PsalC provided discrimination between B. canis isolated in 2005 and others.

• Korean Journal of Pharmacognosy
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• v.49 no.1
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• pp.55-64
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• 2018

Recently, it has been a big issue to distinguish the dried roots of Cynanchum wilfordii and C. auriculatum in health functional food market. The original plant species of Cynanchi Wilfordii Radix belong to the Asclepiadaceae family is differentially described in the national pharmacopoeia of Korea, China and Japan. Owing to the morphological similarities of the dried roots of this plant to those of C. auriculatum, which is often misidentified in Korean herbal medicine marketplace, distinguishing these two species is exceedingly difficult. The purpose of this study was to compare the conventional-PCR with the real-time PCR for detection of C. wilfordii and C. auriculatum DNA. We also tried to realize a quantitative real-time PCR assay using species-specific matK primers, which allowed us to estimate the ratio of C. willfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. The differentiation of intentional and unintentional mixture in this study would be applied to food safety management and can be helpful for protection of consumer's right and cultivators.