• ALGAE
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• v.37 no.1
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• pp.15-32
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• 2022

Inspecting herbaria collections of Laurencia rigida highlighted frequent misidentifications between L. rigida and L. heteroclada f. decussata, two poorly studied taxa from Australia. Recent collections of DNA material, including from topotype material, allowed for re-examination of these two taxa using molecular techniques. Detailed morphological and molecular analyses based on two markers (rbcL and COI-5P) strongly supported these two taxa as being distinct from each other and requiring nomenclatural changes. Comprehensive morphological analyses highlighted features useful for accurate identifications. Interestingly, L. rigida was found to belong to the genus Palisada with evidence from both the morphology and molecular data. Therefore, this study proposed recognizing L. rigida as Palisada rigida comb. nov. Molecular data for L. heteroclada f. decussata on the other hand supported its separation from L. heteroclada, with too great a molecular distance to be considered a variety. Morphological characters that best separated P. rigida from L. decussata included seven characters; number of pericentral cells per vegetative axial segment, the presence of secondary pit connections, the presence of lenticular thickenings, tetrasporangia alignment, the presence of corps en cerise, holdfast morphology, and overall plant shape. Morphologically, L. heteroclada f. decussata was also separated from L. heteroclada, particularly by the following characteristics; ultimate branchlets morphologies, lower order branch lengths, primary axis and holdfast morphologies. Therefore, it was proposed that L. heteroclada f. decussata is recognized at a species level as L. decussata comb. et stat. nov.

• Microbiology and Biotechnology Letters
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• v.41 no.3
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• pp.292-299
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• 2013

In this study, we analyzed the changes in fungal populations of red-pepper fields employing eco-friendly farming methods, such as microbial agents and crop rotation, by using polymerase chain reactions coupled with denaturing gradient gel electrophoresis (PCR-DGGE). Primer specific for fungi were used to determine the contribution of domains to the microbial community. Analysis of planted and non-planted soil samples applying PCR-DGGE technology offered evaluation of long-term patterns in fungal species richness. To evaluate the stability of DGGE patterns from different soils, comparison of planted and non-planted soil samples were compared using PCR-DGGE. The number of DNA fragments obtained from all planted soil samples by DGGE separation was far greater (14 to 15 bands) than that of the non-planted soil samples (3 to 4 bands). In addition, 14 bands were observed from crop continuation soil treated with agrochemicals and 18 bands from crop rotation soil treated with microbial agents. The PCR-DGGE analysis suggests that the use of crop rotation and microbial agents benefits the fungal community more than crop continuation using agrochemicals. These results indicate that crop rotation with microbial agents was better able to support beneficial organisms, enable more effective biological control and maintain a healthier balance of nutrients, organic matter and microorganisms.

• Journal of Life Science
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• v.24 no.7
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• pp.757-761
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• 2014

Olive flounder (Paralichthys olivaceus) is an important aquaculture fish species in Jeju Island, South Korea. Due to the intensification of flounder fish farming, huge amounts of chemical antibiotics are used against several fish diseases. This has many harmful side effects on fish, as well as human consumers. Hence, an alternative to chemical antibiotic agents is needed for disease control. In this study, three strains of rhizobacteria (BRH433-2, TRH415-2, and THJ609-3) were isolated from the rhizosphere of plants. Assays of their antibacterial activity against fish pathogens, such as S. iniae, S. parauberis, V. anguillarum, and E. tarda, were performed with untreated broth culture (without cell separation), supernatant, and precipitated pellets separated by centrifugation. Among these, the cell suspension prepared from the precipitated pellet showed significant antimicrobial activity when compared with that of the untreated broth culture and centrifugal supernatant. These results indicate that the three isolated rhizobacterial strains exhibit antibacterial activity. Analysis of the 16S rDNA sequences of the BRH-433-2, THJ609-3, and TRH415-2 strains showed the highest similarity to Burkholderia gladioli (99.5%), Pseudomonas baetica (97.7%), and P. koreensis and P. baetica (98.4%), respectively. We suggest that the strains hold promise in disease management of fish.

• Food Engineering Progress
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• v.15 no.4
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• pp.370-375
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• 2011

In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monoytogenes, Staphyloocus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over $10^{5}$ CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over $10^{1}$ CFU/g, but only those of B. cereus were over $10^{3}$ CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1971-1982
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• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.372-378
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• 2017

For comparison of the transcription profiles in apple (Malus domestica L.) cultivars differing in flesh color expression, two cDNA libraries were constructed. Differences in gene expression between red flesh apple cultivar, 'Redfield' and white flesh apple cultivar, 'Granny Smith' were investigated by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the red flesh apple cultivar and white flesh apple cultivar were selected for nucleotide sequence determination and homology searches. High resolution melting (HRM) technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between red flesh apple cultivars and white flesh apple cultivars. We applied high resolution melting (HRM) analysis to discover single nucleotide polymorphisms (SNP) based on the predicted SNP information derived from the apple EST database. All 103 pairs of SNPs were discriminated, and the HRM profiles of amplicons were established. Putative SNPs were screened from the apple EST contigs by HRM analysis displayed specific difference between 10 red flesh apple cultivars and 11 white flesh apple cultivars. In this study, we report an efficient method to develop SNP markers from an EST database with HRM analysis in apple. These SNP markers could be useful for apple marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in apple cultivars.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1107-1112
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• 2009

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

• The Korean Journal of Nuclear Medicine Technology
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• v.12 no.1
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• pp.62-65
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• 2008

Purpose: Vitamin $B_{12}$ and folic acid are type of water-soluble vitamin and those work as a secondary vital enzyme, but especially those are involved in the nuclear DNA synthesis. Those are mainly measured in order to diagnose megaloblastic anemia and to assess the storage of folic acid during pregnancy. It is generally known that the hemolyzed serum is useless for folic acid and vitamin $B_{12}$, but it is not easy to abide by this information because our lab (Samkwang Medical Laboratories) is reference laboratory. We tested how much the extent of hemolyzed serum had influence on the results. Materials and Method: We performed the tests of vitamin $B_{12}$, folic acid. For the hemolysis effect study, we used 40 cases. According to the level of A, B and C groups, each group had 10 samples which were mechanically hemolyzed serum. Results: Hemolysis did not affect the vitamin $B_{12}$ results. However in case of folic acid, the value increased according to the degree of hemolysis. And severe hemolyzed cases had the highest value (greater than 20 ng/ml). Conclusions: Preventing the hemolysis, it has to be informed that blood-collecting, separation and storage are performed more carefully. As you see from the above results, hemolyzed serum is not proper for folic acid test, and hemolysis does not affect the results of vitamin $B_{12}$. However, for the more accurate results, it is necessary to avoid hemolysis.

• 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.

• Journal of Korean Medicine for Obesity Research
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• v.19 no.2
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• pp.97-105
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• 2019

Objectives: We investigated differences between the tracheostomized and the non-tracheostomized stroke patients through microbiological analysis for the purpose of preliminary explorations of full-scale clinical research in the future. Methods: We collected tracheal aspirates samples from 5 stroke patients with tracheostomy and expectorated sputum samples from 5 stroke patients without tracheostomy. Genomic DNA from sputum samples was isolated using QIAamp DNA mini kit. The sequences were processed using Quantitative Insights into Microbial Ecology 1.9.0. Alpha-diversity was calculated using the Chao1 estimator. Beta-diversity was analyzed by UniFrac-based principal coordinates analysis (PCoA). To confirm taxa with different abundance among the groups, linear discriminant analysis effect size analysis was performed. Results: Although alpha-diversity value of the tracheostomized group was higher than that of the non-tracheostomized group, there was no statistically significant difference. In PCoA, clear separation was seen between clusters of the tracheostomized group and that of the non-tracheostomized group. In both groups, Bacteroidetes, Proteobacteria, Fusobacteria, Firmicutes, Actinobacteria were identified as dominant in phylum level. In particular, relative richness of Proteobacteria was found to be 31% more in the tracheotomized group (36.6%) than the non-tracheostomized group (5.6%)(P<0.05). In genus level, Neisseria (24%), Prevotella (17%), Streptococcus (13%), Fusobacteria (11%), Porphyromonas (7%) were identified as dominant in the tracheostomized group. In the non-tracheostomized group, Prevotella (38%), Veillonella (20%), Neisseria (9%) were genera that found to be dominant. Conclusions: It is meaningful in that the tracheostomized group has been identified a higher rate of microbiotas known as pathogenic in respiratory diseases compared to the non-tracheostomized group, confirming the possibility that the risk of opportunity infection may be higher.