• The Korea Journal of Herbology
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• v.30 no.3
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• pp.41-47
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• 2015

Objectives : Due to the morphological similarity of the pericarp and description of multi-species in National Pharmacopoeia of Korea and China, the Zanthoxylum Pericarpium is difficult to authenticate adulterant in species levels. Therefore, we introduced the sequence analysis of DNA barcode and identification of single nucleotide polymorphism(SNP) to establish a reliable tool for the distinction of Zanthoxylum Pericarpium from its adulterants. Methods : To analyze DNA barcode region, genomic DNA was extracted from twenty-four specimens of authentic Zanthoxylum species and inauthentic adulterant and the individual internal transcribed spacer regions (rDNA-ITS and ITS2) of nuclear ribosomal RNA gene were amplified using ITS1, ITS2-S2F, and ITS4 primer. For identification of species-specific sequences, a comparative analysis was performed using entire DNA barcode sequences. Results : In comparison of four Zanthoxylum ITS2 sequences, we identified 16, 4, 6, and 4 distinct species-specific nucleotides enough to distinguish Z. schinifolium, Z. bungeanum, Z. piperitum, and Z. simulans, respectively. The sequence differences were available genetic marker to discriminate four species. Futhermore, phylogenetic relationship revealed a clear classification between different Zanthoxylum species showing 4 different clusters. These results indicated that comparative analysis of ITS2 DNA barcode was an useful genetic marker to authenticate Zanthoxylum Pericarpium in species levels. Conclusions : The marker nucleotides, enough to distinguish Z. schinifolium, Z. piperitum, Z. bungeanum, and Z. simulans, were obtained at 30 SNP marker nucleotides from ITS2 sequences. These differences could be used to authenticate official Zanthoxylum Pericarpium from its adulterants as well as discriminating each four species.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.169-175
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• 2018

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

• Journal of Plant Biotechnology
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• v.47 no.4
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• pp.309-315
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• 2020

Advances in biotechnology have led to progress in crop genetic engineering to improve agricultural productivity. The use of genetically modified (GM) crops has increased, as have consumers' and regulators' concerns about the safety of GM crops to human health, and ecological biodiversity. As such, the identification of GM crops is a critical issue for developers and distributors, and their labeling is mandatory. Multiplex polymerase chain reaction (PCR) has been developed and its use validated for the detection and identification of GM crops in quarantine. Herein, we established a simultaneous detection method to identify four GM maize events. Event-specific primers were designed between the junction region of transgene and genome of four GM maize lines, namely 5307, DAS-40278-9, MON87460, and MON87427. To verify the efficiency and accuracy of the multiplex PCR we used specificity analysis, limit of detection evaluation, and mixed certified reference materials identification. The multiplex PCR method was applied to analyze 29 living, modified maize volunteers collected in South Korea in 2018 and 2019. We performed multiplex PCR analysis to identify events and confirmed the result by simplex PCR using each event-specific primer. As a result, rather than detecting each event individually, the simultaneous detection PCR method enabled the rapid analysis of 29 GM maize volunteers. Thus, the novel multiplex PCR method is applicable for living modified organism volunteer identification.

• The Plant Pathology Journal
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• v.39 no.3
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• pp.255-264
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• 2023

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus Mastrevirus (family Geminiviridae) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into Nicotiana benthamiana using three Agrobacterium tumefaciens strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The A. tumefaciens strain LBA4404 was the most effective at transferring the SPSMV-1 genome to N. benthamiana. We confirmed the viral replication in N. benthamiana samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

• Korean Journal of Medicinal Crop Science
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• v.23 no.6
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• pp.439-445
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• 2015

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.

• Korean Journal of Veterinary Research
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• v.38 no.3
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• pp.565-573
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• 1998

To study the specific tools for the diagnosis of Newcastle disease virus (NDV) in chicken, polymerase chain reaction (PCR) and its presumable conditions were evaluated for the detection of hemagglutinin-neuraminidase (HN) gene of NDV RNA. For these purposes, Kyojeongwon strain of the NDV was propagated in allantoic cavity of SPF embryonating chicken eggs, and viral RNA was extracted from fractionated virus after the allantoic fluids were ultracentrifuged with sucrose gradient. The first-strand cDNA was then made for the HN gene of NDV RNA by reverse transcription at $42^{\circ}C$ for 1 hour using specific primer complementary to the HN gene. The single-stranded cDNA was used as template in the PCR of the HN-DNA, and various conditions of the PCR were evaluated to set up method for the specific detection of the HN-DNA. The PCR conditions promising for the detection of HN gene consist of preheating at $94^{\circ}C$, 5 min, 30 cycles of denaturation at $94^{\circ}C$, 1 min, annealing at $55^{\circ}C$, 1 min and polymerization at $72^{\circ}C$, 2 min, and a cycle of extension at $72^{\circ}C$, 5 min. when NDVs of allantoic fluids without fractionation were applied to the above PCR condition, the HN genes were detected effectively not only from Kyojeongwon but from other velogenic strains such as Herts and a field isolate.

• Genomics & Informatics
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• v.21 no.4
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• ALGAE
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• v.33 no.1
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• pp.21-35
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• 2018

The phototrophic dinoflagellate genus Scrippsiella is known to have a worldwide distribution. Here, we report for the first time, the occurrence of Scrippsiella lachrymosa in Korean waters. Unlike the other stains of S. lachrymosa whose cultures had been established from cysts in the sediments, the clonal culture of the Korean strain of S. lachrymosa was established from motile cells. When the sulcal plates of S. lachrymosa, which have not been fully described to date, were carefully examined using scanning electron microscopy, the Korean strain of S. lachrymosa clearly exhibited the anterior sulcal plate (s.a.), right sulcal plate (s.d.), left sulcal plate (s.s.), median sulcal plate (s.m.), and posterior sulcal plate (s.p.). When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of S. lachrymosa was ca. 1% different from those of two Norwegian strains of S. lachrymosa, the only strains for which LSU sequences have been reported. The internal transcribed spacer (ITS) rDNA sequence of the Korean strain of S. lachrymosa was also ca. 1% different from those of the Scottish and Chinese strains and 3% different from those of the Canadian, German, Greek, and Portuguese strains. Thus, the Korean S. lachrymosa strain has unique LSU and ITS sequences. The abundances of S. lachrymosa in the waters of 28 stations, located in the East, West, and South Sea of Korea, were quantified in four seasons from January 2016 to October 2017, using quantitative real-time polymerase chain reaction method and newly designed specific primer-probe sets. Its abundances were >$0.1cells\;mL^{-1}$ at eight stations in January and March 2016 and March 2017, and its highest abundance in Korean waters was $26cells\;mL^{-1}$. Thus, S. lachrymosa has a nationwide distribution in Korean waters as motile cells.

• Korean journal of applied entomology
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• v.42 no.1
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• pp.65-70
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• 2003

For the purpose of the protection of beneficial insects from pathogens and the development of control agent against pests, a strain of Metarhizium sp. was isolated from the infected Protaetia brevitarsis seulensis larvae in Korea. Under the scanning electron microscope, the isolate, Metarhizium sp. KMA-1, showed distinct formation of conidia on the palisade-like masse which were comprised of elongate chains and this shape is a typical feature of Metarhizium species. PCR techniques were used to identify the isolate and the primers used were designed on the basis of two kinds of rRNAs sequences, 28S rRNA and internal transcribed spacer(ITS). The specific PCR products from each primer set were amplified and the DNA sequences were determined for the similarity comparison. Sequence alignment of these fragments using GenBank database resulted in the highest homology similarity between the isolate Metarhizium sp. KMA-1 and M. anisopliae. From these results, the isolate Metarhizium sp. KMA-1 in this study was identified as M. anisopliae.

• 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.