• Toxicological Research
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• v.32 no.1
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.244-247
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• 2004

To investigate the effects of elevated $CO_2$ on the denitrifying bacterial community structure in a wetland soil, dynamics of bacterial community structure was explored in an artificial wetland ecosystem with one of three plant species (T. latifolia, S. lacustris, and 1. effusus) under two levels of $CO_2$(370 ppm or 740 ppm) after 110day incubation. For the analysis of bacterial community structure, functional genes such as nitrite reductase genes (nirS) were PCR-amplified followed by cloning of PCR products and screening by restriction fragment length polymorphism (RFLP). nirS gene fragments were amplified in all analyzed soil samples. Species richness estimated by the number of distinct phylotypes were 83 and 95 in the ambient $CO_2$ treatment and the elevated treatment, respectively. Two phylotypes (type 1 and type 2) were dominant in both of the treatments. Elevated $CO_2$ treatment increased species richness of denitrifying as well as changed a large proportion of denitrifier phylotypes compared to those of the ambient treatment. Overall, the data in this study suggested that the denitrifying communities in the wetland soil are diverse and that the richness of denitrifying bacterial community might be affected by elevated $CO_2$ treatment.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.18-27
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• 2020

The correct distinction of carbapenem-resistant Enterobacteriaceae (CRE) and ccarbapenemase producing Enterobacteriaceae (CPE) and the rapid detection of CPE are important for instituting the correct treatment and management of clinical infections. Screening protocols are mainly based on cultures of rectal swab specimens on selective media followed by phenotypic tests to confirm a carbapenem-hydrolyzing activity, the rapid carbapenem inactivation method, lateral flow immunoassay, the matrix-assisted laser desorption ionization-time-of-flight test and molecular methods. The CPE is accurate for detection, and is essential for the clinical treatment and prevention of infections. A variety of phenotypic methods and gene-based methods are available for the rapid detection of carbapenemases, and these are expected to be routinely used in clinical microbiology laboratories. Therefore, to control the spread of carbapenemase, many laboratories around the world will need to use reliable, fast, high efficiency, simple and low cost methods. Optimal effects in patient applications would require rapid testing of CRE to provide reproducible support for antimicrobial management interventions or the treatment by various types of clinicians. For the optimal test method, it is necessary to combine complementary test methods to discriminate between various resistant bacterial species and to discover the genetic diversity of various types of carbapenemase for arriving at the best infection control strategy.

• Journal of Life Science
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• v.27 no.10
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• pp.1161-1167
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• 2017

In order to find a new antimicrobial bacterium, we performed screening for antimicrobial activity of bacteria isolated from the eggs of a sea hare. The newly identified strain was designated as Phaeobacter inhibens KJ-2, based on the biochemical characterization and 16S rRNA gene sequence analysis. A colony of P. inhibens KJ-2 showed a circular and ruler-like smooth form at the edge, and a brown color. However, when maintained with a longer incubation time, its coloring was transformed into dark brown. From the result of SEM, P. inhibens KJ-2 is a bacillus which has a length of $0.8{\sim}1.0{\mu}m$ and a width of $0.4{\sim}0.6{\mu}m$. The optimal growth and antimicrobial activity were observed by shaking the culture for 24 hr at $20^{\circ}C$, which showed potent activity against pathogenic bacteria including Vibrio logei, Vibrio campbellii, Vibrio mimicus, Vibrio vulnificus, and Vibrio salmonicida. The antimicrobial activity was proportional to the amount of produced acylated homoserine lactones (AHLs). Therefore, we suggest that production of antimicrobial materials from P. inhibens KJ-2 is regulated by Quorum sensing (QS).

• Molecules and Cells
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• v.25 no.3
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• pp.407-416
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• 2008

Thirteen near-isogenic lines (NILs) of japonica rice were developed via a backcross method using the recurrent parent Chucheong, which is of good eating quality but is susceptible to Magnaporthe grisea, and three blast resistant japonica donors, Seolak, Daeseong and Bongkwang. The agro-morphological traits of these NILs, such as heading date, culm length, and panicle length, were similar to those of Chucheong. In a genome-wide scan using 158 SSR markers, chromosome segments of Chucheong were identified in most polymorphic regions of the 13 NIL plants, and only a few chromosome segments were found to have been substituted by donor alleles. The genetic similarities of the 13 NILs to the recurrent parent Chucheong averaged 0.961, with a range of 0.932-0.984. Analysis of 13 major blast resistance (R) genes in these lines using specific DNA markers showed that each NIL appeared to contain some combination of the four R genes, Pib, Pii, Pik-m and Pita-2, with the first three genes being present in each line. Screening of nine M. grisea isolates revealed that one NIL M7 was resistant to all nine isolates; the remaining NILs were each resistant to between three and seven isolates, except for NIL M106, which was resistant to only two isolates. In a blast nursery experiment, all the NILs proved to be more resistant than Chucheong. These newly developed NILs have potential as commercial rice varieties because of their increased resistance to M. grisea combined with the desirable agronomic traits of Chucheong. They also provide material for studying the genetic basis of blast resistance.

• Molecules and Cells
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• v.38 no.4
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• pp.373-379
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• 2015

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of $4.92{\mu}M$, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.721-727
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• 2007

Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase(L-TA) from Streptomyces coelicolor A3(2)(SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and F18I in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at $60^{\circ}C$ was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at $63^{\circ}C$ was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine(L-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.

• Korean Journal of Food Science and Technology
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• v.45 no.1
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• pp.133-136
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• 2013

The purpose of this study was to compare a conventional culture method and real-time PCR for the detection of Yersinia enterocolitica (Y. enterocolitica) in sausage and in vegetable salad. Food samples inoculated with Y. enterocolitica were enriched in peptone-sorbitol bile-broth, and swabs were then streaked onto cefsulodin-irgasan-novobiocin agar. Biochemical tests for suspected colonies were performed with an API 20E strip. In parallel, real-time PCR was performed, targeting the 16S rRNA gene using 1 mL of enrichment broth. In sausage, the number of positive samples detected by culture method (49 out of 60) was similar (p>0.05) with that of real-time PCR (50 out of 60). However, the number of positive samples of real-time PCR (26 out of 60) was significantly higher (p<0.05) than that of the conventional culture method (6 out of 60) in vegetable salad. Real-time PCR could be an effective screening tool for detecting Y. enterocolitica, particularly in food samples with high levels of background flora, such as a vegetable salad.

• Journal of Chitin and Chitosan
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• v.23 no.4
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• pp.228-233
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• 2018

We isolated microorganisms from soil, which is sampled at forest, Kyeonbuk, Korea, as cellulolytic microorganisms. The isolated strains were identified by analysis of 16S rRNA gene from the starins. The result, four kinds of Bacillus subtilis, one kind of Bacillus amyloliquefaciens, and one kind of Bacillus cereus were identified. Among these strains, Bacillus subtilis was selected due to its high cellulase activity and this strain was named as Bacillus subtilis CNS. The optimum pH and temperature of the cellulase from Bacillus subtilis CNS was pH 5.0 and $40^{\circ}C$, respectively. In the investigation of pH and temperature stability, the cellulase from Bacillus subtilis NSC stabled pH 4.0~6.0 range and until $40^{\circ}C$ for 30 min perfectly. In the enzyme activity for various cellulosic substrate, cellulase from Bacillus subtilis CNS showed the highest activity for CM-cellulose. And, the enzyme activities for alkali swollen cellulose, Alpha-cellulose, Sigmacell-cellulose, and Avicel were approximately 31%, 8%, 8% and 4% of activity for CM-cellulose, respectively. In the degradation of CM-cellulose, the 0.26 U/ml and 0.52 U/ml of cellulase showed 0.43 and 0.76 U/ml activity for CM-cellulose after the reaction of 120 min, respectively.

• Journal of Food Hygiene and Safety
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• v.36 no.5
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• pp.407-417
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• 2021

In order to investigate frequency of fungal contamination in ginseng sprout, we collected 18 types of retail ginseng sprouts and analyzed them. Overall frequency of fungal contamination ranged from 113.3 to 174.1% with the highest occurrence of Penicillium spp. Fungal detection rate was significantly higher in moss than in stem, leaf and root of ginseng sprout. Penicillium spp. occurred in leaf and stem with the highest incidence and Fusarium spp., in root. Among Penicillium spp. and Fusarium spp., P. olsonii and F. oxysporum were dominant, respectively. Nine Fusarium species, Aspergillus westerdijkiae, Aspergillus flavus, and 11 Penicillium species were identified by phylogenetic analysis. PCR screening of mycotoxigenic potential revealed that 19 out of 25 isolates tested were positive for respective mycotoxin biosynthetic gene. Two 2 A. flavus and 11 A. westerdijkiae isolates produced varying amount of aflatoxin or ochratoxin A in czapek yeast extract brothsome of which showed high levels of mycotoxin production. These results suggests a need for continuous monitoring and management program to control fungal contamination in the ginseng sprout production chain.