• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.88-93
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• 2016

Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

• Journal of Life Science
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• v.28 no.9
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• pp.1062-1067
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• 2018

Reliable labeling of fish products can reassure consumers regarding the identity and quality of seafoods. Therefore, techniques that can identify adulteration or mislabeling are valuable. To rapidly identify two Trachurus species, Trachurus japonicus and Trachurus novaezelandiae, a highly efficient, rapid, duplex polymerase chain reaction (PCR) having two species-specific primers simultaneously was identified. This species-specific primer focused on a single nucleotide mismatch in the 3'-terminal base of a primer designed in the mitochondrial cytochrome c oxidase (COI) subunit I DNA. To optimize the duplex PCR condition, gradient PCR reactions were conducted to determine the primer annealing temperature and the primer concentration. The PCR's product was observed on the gel, suggesting that DNA molecules may be useful in differentiating the two species. The length of the amplification fragments were 103 bp for Trachurus japonicus and 214 bp for Trachurus novaezelandiae, which, along with the species-specific primer visualized by agarose gel electrophoresis, enabled accurate distinction of the species of the Trachurus genus. These results indicate that the duplex PCR, which has a species-specific primer based on single nucleotide polymorphism (SNP), can be useful for rapidly differentiating the two species of Trachurus. This duplex PCR analysis is simple, rapid, and reliable, and could be beneficial to protecting consumers' rights.

• The Korea Journal of Herbology
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• v.34 no.5
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• pp.13-20
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• 2019

Objectives : To ensure the safety, quality and pharmacological efficacy of Batryticatus Bombyx, it is important to discriminate with adulterants. In Korean Herbal Pharmacopoeias (KHP), the authentic species of Batryticatus Bombyx is defined only Bombyx mori. Therefore, the aim of this study is establishment of PCR assay method using the sequence characterized amplified region (SCAR) marker based on COI DNA barcode for discriminating six species related to Batryticatus Bombyx. Methods : Seventeen samples of six species (Bombyx mori, Bombyx mandarina, Rhodinia fugax, Oberthueria caeca, Actias artemis, and Caligula japponica) were collected from different habitate and nucleotide sequences of cytochrome c oxidase subunit I(COI) barcode regions were analyzed by Sanger sequencing methods. To develop SCAR-based PCR assay method, we designed species-specific primers based on COI sequence variabilities and verified those specificities using 17 samples of six species as well as commercial herbal medicines. Results : In comparative multiple analysis of COI sequences, six species were distinguished by species-specific nucleotides at the species level. To develop rapid and reliable PCR assay method for genetic authentication of Batryticatus Bombyx, therefore, we designed species-specific SCAR primers based on these nucleotide sequences and confirmed those specificities. Using these SCAR primers, We also established simple conventional PCR assay method using these SCAR primers at the species level. Conclusions : The comparative analysis of COI sequences and SCAR-based PCR assay methods represented equal results for distinguishing authentic Batryticatus Bombyx and adulterations at the species level. Therefore, our results are expected protecting adulteration of herbal medicine Batryticatus Bombyx.

• Korean Journal of Ecology and Environment
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• v.55 no.4
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• pp.352-359
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• 2022

Recently, with the development of genetic technology, interest in environmental DNA (eDNA) to study biodiversity according to molecular biological approaches is increasing. Environmental DNA has many advantages over traditional research methods for biological communities distributed in the environment but highly depends on the established base sequence database. This study conducted a comprehensive analysis of the habitat status and classification at the genus level, which is mainly used in eDNA (12S rRNA, 16S rRNA, 18S rRNA, COI, and CYTB), focusing on Korean registration taxon groups (phytoplankton, zooplankton, macroinvertebrates, and fish). As a result, phytoplankton and zooplankton showed the highest taxa proportion in 18S rRNA, and macroinvertebrates observed the highest ratio in the nucleotide sequence database in COI. In fish, all genes except 18S rRNA showed a high taxon ratio. Based on the Korean registration taxon group, the gene construction of the top 20 genera according to bio density observed that most of the phytoplankton were registered in 18S rRNA, and the most significant number of COI nucleotide sequences were established in macroinvertebrates. In addition, it was confirmed that there is a nucleotide sequence for the top 20 genera in 12S rRNA, 16S rRNA, and CYTB in fish. These results provided comprehensive information on the genes suitable for eDNA research for each taxon group.

• Korean Journal of Ichthyology
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• v.36 no.1
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• pp.94-100
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• 2024

During ichthyoplankton survey in the southwestern sea of Korea, we collected six individuals of noodlefish larvae and juveniles between April and May 2023. They were identified as Neosalanx anderssoni by mitochondrial DNA cytochrome c oxidase subunit I or 16S ribosomal RNA sequences, and their external morphological traits were described for the first time. All six individuals have a slender and elongated body. When preflexion and flexion larval stages (10.24 mm notochord length, NL and 15.47 mm total length, TL, respectively), oval-shaped black melanophores were distributed in a row along the ventral side of the gut. However, when postflexion larval and juvenile stages (23.58~25.90 mm TL, and 29.20~31.26 mm TL, respectively), melanophores on the ventral side of the gut were disappeared, and dark spot-shaped melanophores appeared along the dorsal side of the gut in a single row. Also, from the postflexion larval stage (23.58 mm TL), two large black spots began to appear symmetrically on the caudal fin. Our results suggest that N. anderssoni may use coastal area as spawning and/or nursery ground unlike previous study (Kim and Park, 2002).

• Journal of Food Hygiene and Safety
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• v.29 no.4
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• pp.347-355
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• 2014

In this study, the detection method was developed using molecular biological technique to distinguish authenticity of animal raw materials. The genes for distinction of species about animals targeted at Cytochrome c oxidase subunit I (COI), Cytochrome b (Cytb), and 16S ribosomal RNA (16S rRNA) genes in mitochondrial DNA. The species-specific primers were designed by that Polymerase Chain Reaction (PCR) product size was around 200 bp for applying to processed products. The target 24 raw materials were 2 species of domestic animals, 6 species of poultry, 2 species of freshwater fishes, 13 species of marine fishes and 1 species of crustaceans. The results of PCR for Rabbit, Fox, Pheasant, Domestic Pigeon, Rufous Turtle Dove, Quail, Tree Sparrow, Barn Swallow, Catfish, Mandarin Fish, Flying Fish, Mallotus villosus, Pacific Herring, Sand Lance, Japanese Anchovy, Small Yellow Croaker, Halibut, Jacopever, Skate Ray, Ray, File Fish, Sea Bass, Sea Urchin, and Lobster raw materials were confirmed 113 bp ~ 218 bp, respectively. Also, non-specific PCR products were not detected in compare species by species-specific primers. The method using primers developed in this study may be applied to distinguish an authenticity of food materials included animal raw materials for various processed products.

• Korean journal of applied entomology
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• v.59 no.2
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• pp.93-107
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• 2020

Adults of seven noctuid potential pests (Spodoptera frugiperda, S. litura, S. exigua, Ctenoplusia agnata, Mythimna loreyi, Athetis dissimilis, and A. lepigone) of soybean and maize in Suwon, Korea were identified by their morphological characteristics in the wing pattern and male genitalia and partial mitochondrial DNA sequences of cytochrome c oxidase subunit 1 gene. The generation number of adults that emerge annually in six species (except A. lepigone) was estimated from the data on density fluctuations of adults caught in sex pheromone traps in 2019 and the forecasted data using temperature-associated development and reproduction models for those species. S. frugiperda adults were caught from July 27th to October 31st in 2019, and hence were initially estimated to emerge three times per year. But, it was finally expected that S. frugiperda adults could possibly emerge a total of four times per year in Suwon, considering larval emergence observed during mid- and late June in other areas. Adult emergence of S. litura, S. exigua, C. agnata, and M. loreyi in 2019 was observed from May 29th to November 6th, from May 14th to November 6th, from May 26th to October 25th, and from May 31st to November 23rd, respectively. Annual adult emergence of these four species was estimated as at least four times. Adults of A. dissimilis were caught from May 26th to September 11th in 2019, and adult emergence was estimated at only twice per annum. It was postulated that the first adult populations of five species except the two Athetis species were probably migrated from other areas.