• Asian-Australasian Journal of Animal Sciences
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• 제18권11호
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• pp.1513-1518
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• 2005

The work reported here is an attempt to explore the possibility of DNA microsatellite loci transfer (cross-species amplification) to other economically important aquacultured catfish species other than its source species. A total of 25 new microsatellite loci developed for riverine catfish, Mystus nemurus were successfully cross-amplified in two distantly related catfish species within the suborder Siluroidei. Five out of the 19 loci that successfully cross-amplified in Pangasius micronemus were polymorphic, while for Clarias batrachus, cross-amplification was successful using 17 polymorphic loci. The observed heterozygosities were high for all the three catfishes. The results indicated that microsatellite loci could be as polymorphic in non-source species as in the source species.

• 식물분류학회지
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• 제50권3호
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• pp.327-332
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• 2020

Microsatellite markers were developed as a tool for phylogeographic studies of Hosta capitata. We also assessed cross-amplification in species closely related to Hosta capitata. We produced 28 polymorphic microsatellite markers by mapping 300 bp paired-end reads obtained from Illumina MiSeq data of H. capitata. In H. capitata, the number of alleles per locus ranged from 1 to 13. Observed and expected heterozygosity ranged from 0.000 to 0.844 and 0.000 to 0.832, respectively. Additionally, 13 loci were successfully transferable to the related species of H. minor and H. venusta. These markers will provide a powerful genetic tool not only for elucidating the phylogeographic patterns of H. capitata populations but also for studying the genetic delimitation of H. capitata from its related species.

• 식물병연구
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• 제27권1호
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• pp.38-44
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• 2021

Xylella fastidiosa is the most damaging pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, the loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay were developed to mqsA gene of citrate-synthase (XF 1535) X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than current published tests for detection of X. fastidiosa when screened against 16 isolates representing the four major subgroups of the bacterium from a range of host species. No cross reaction with DNA from healthy hosts or other species of bacteria has been observed. The LAMP and PCR assays could detect 10-4 pmol and 100 copies of the gene, respectively. Hydroxynaphthol blue was evaluated as an endpoint detection method for LAMP. There was a significant color shift that signaled the existence of the bacterium when at least 100 copies of the target template were present.

• 생명과학회지
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• 제27권7호
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• pp.746-753
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• 2017

참조기는 민어과에 속하는 우리나라의 중요한 산업 어종 중 하나이다. 최근 과도한 남획과 해양 환경의 변화로 참조기의 어획량이 줄어들자 일부 유통과정에서 유사어종인 부세, 흑조기 및 긴가이석태를 참조기로 둔갑시키는 사례가 빈번하게 발생하고 있다. 이에 본 연구에서는 종 특이 primer를 사용하여 참조기, 부세, 흑조기 및 긴가이석태를 신속하게 분석할 수 있는 방법을 마련하였다. 약 1,400 bp의 미토콘드리아 COI 유전자 분석을 통하여 종간 특이성을 나타내는 단일염기다형성 유전자를 탐색하였으며, PCR 증폭산물의 크기를 고려하여 4개의 종특이적 정방향 primer를 제작하였다. 단일 PCR을 이용한 종간 교차반응을 통하여 최적의 PCR 조건을 확립하였으며, 이후 제작된 4개의 정방향 primer를 혼합하여 4종에 대한 다중 PCR 반응을 진행하였다. 증폭된 산물은 전기영동을 통해 크기에 따라 1,540 bp, 1,013 bp, 470 bp 그리고 182 bp로 분리되었으며, 각각 참조기, 흑조기, 부세, 긴가이석태로 명확하게 판별이 가능하였다. PCR 민감도 측정에서도 모든 종에서 $0.1ng/{\mu}l$의 농도까지 검출 가능함을 확인하였다. 따라서, 본 연구에서 개발된 참조기와 유사어종에 대한 종특이 다중 PCR 분석법은 정확도와 민감도가 우수하여, 불법 유통가능성이 있는 제품에 대한 신속하고 정확한 판별로 식품안전관리에 효과적으로 활용될 것이라 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1046-1048
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• 2003

A polymerase chain reaction (PCR) assay was developed to differentiate buffalo meat from the meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon sambhur (Cervus unicolor unicolor), cattle (Bovine), goat (Caprine), pig (Porcine), and sheep (Ovine). A set of primers were designed according to the sequence of the mitochondrial cytochrome b gene of bubalus bubalis and by PCR amplification a band of approximately 242 bp band was observed with buffalo DNA. These primers did not cross-react with DNA of other animal species tested in the study under the specified reaction conditions. A band of 649 bp was observed for all animal species tested when DNA was amplified with the universal primers indicating the presence of mitochondrial DNA in the samples. The technique was sensitive enough to identify rotten (10 days post slaughter), dried and cooked buffalo meat. The absence of a cross reaction with human DNA using the buffalo specific primers eliminates possible false positive reactions.

• 식물분류학회지
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• 제50권1호
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• pp.22-26
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• 2020

Chrysosplenium aureobracteatum Y. I. Kim & Y. D. Kim (Saxifragaceae) is a recently described endemic species growing in the central part of the Korean peninsula. It requires constant monitoring for conservation due to its limited distributions. There is also a need for molecular markers for proper assessments of the genetic differentiation of C. aureobracteatum from species morphologically similar to it. In this study, we developed microsatellite markers that can be used to evaluate the genetic diversity of this species, representing fundamental data with which to conserve the natural populations of the species. A total of 17 expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed by the Illumina pair-end sequencing of the transcriptomes of C. aureobracteatum. These markers were successfully applied to populations of C. aureobracteatum and to its most closely related species, C. barbatum, revealing high polymorphism in both species. The EST-SSR markers developed in this study were proven to be useful not only to monitor the population genetic structure of C. aureobracteatum for conservation purposes but also to study the genetic delimitation of the species from species closely related to it.

• Animal Systematics, Evolution and Diversity
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• 제33권2호
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• pp.136-139
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• 2017

The genus Iksookimia (Actinopterygii: Cypriniformes: Cobitidae) is a bottom-dwelling freshwater loaches, which are well-known as their endemism and high geographic variation. However, population genetic relationships among Iksookimia spp. have remained unclear due to a shortage of genetic markers that can be applied generally in the genus. Here, we developed high-resolving microsatellite markers using I. koreensis and I. longicorpa as representatives of Iksookimia species because of their wide distribution range and phylogenetic position. Ten of polymorphic microsatellite loci were isolated from Iksookimia koreensis and were successfully cross-amplified in I. longicorpa. The mean number of observed alleles per locus was about 10.4 (range, 2-17) for I. koreensis and about 13.2 (range, 2-24) for I. longicorpa. The loci, IK03 and IK08, deviated from the Hardy-Weinberg equilibrium in I. koreensis, after applying the Bonferroni correction. The microsatellite markers obtained in the present study will be useful to evaluate population genetic structure and to establish conservation strategies for I. koreensis and related Iksookimia species.

• 한국축산식품학회지
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• 제34권6호
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• pp.799-807
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• 2014

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be $85.56{\pm}0.07^{\circ}C$ for cattle, $84.96{\pm}0.08^{\circ}C$ for pig, and $85.99{\pm}0.05^{\circ}C$ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); $84.91{\pm}0.11^{\circ}C$ for goat and $83.90{\pm}0.11^{\circ}C$ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and $86.31{\pm}0.23^{\circ}C$ for chicken, $88.66{\pm}0.12^{\circ}C$ for duck, and $84.49{\pm}0.08^{\circ}C$ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from $10pg/{\mu}L$ to $100fg/{\mu}L$ levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

• Journal of Microbiology
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• 제44권1호
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1672-1683
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• 2021

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla_CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla_CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg²⁺, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63℃ for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10^-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.