• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.732-739
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• 2014

We describe the development of a polymerase chain reaction method for the rapid, precise, and specific detection of the Xanthomonas oryzae pv. oryzae (Xoo) K3a race, the bacterial blight pathogen of rice. The specific primer set was designed to amplify a genomic locus derived from an amplified fragment length polymorphism specific for the K3a race. The 1,024 bp amplicon was generated from the DNA of 13 isolates of Xoo K3a races out of 119 isolates of other races, pathovars, and Xanthomonas species. The assay does not require isolated bacterial cells or DNA extraction. Moreover, the pathogen was quickly detected in rice leaf 2 days after inoculation with bacteria and at a distance of 8 cm from the rice leaf 5 days later. The results suggest that this PCR-based assay will be a useful and powerful tool for the detection and identification of the Xoo K3a race in rice plants as well as for early diagnosis of infection in paddy fields.

• 농업과학연구
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• 제40권2호
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• pp.115-121
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• 2013

Polymorphic bands of two fine-leaf zoysiagrass mutants (CNU 70-1, CNU 70-2) induced via a gamma-ray irradiation on seeds of Zoysia japonica were obtained by using randomly amplified polymorphic DNA (RAPD) primers. The genotype-specific fragments were then converted into PCR-based sequence characterized amplified region (SCAR) markers, which are now amenable to detecting them among other zoysiagrass species widely noticeable in Korea. The CNU 70-1-specific primer set amplified about 900 bp successfully, while the CNU 70-6 marker produced the expected 1,500 bp band, by which those markers were nominated by CNU 70-1_900 and CNU 70-6_1500 SCARs, respectively. The developed SCAR markers can be an applicable tool in sod industry where illegal appropriation hampers breeder's right and profits due to the turfgrass plant vegetatively propagating.

• 한국어병학회지
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• 제34권2호
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• pp.133-140
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• 2021

With the recent isolation of a new betanodavirus in shellfish, Korean Shellfish Nervous Necrosis Virus (KSNNV), it has also been identified the reassortant KSNNV of two RNA segments, in which one segment is KSNNV genotype but the other one is known genotype. In this study, we confirmed that the ressortant KSNNVs obtained in previous screening study of our laboratory for betanodaviruses in shellfish were KS/RGNNV and RG/KSNNV type by performing two consecutive multiplex RT-PCR on each RNA1 and RNA2 segment (R1- and R2-discriminative multiplex two-step RT-PCR, respectively) to determine the genotype of each segment based on the size of amplicon. In the pathogenicity analysis, none of the reassortants induced specific external symptoms or mortality of VNN, but viruses of 2 × 10⁴~10⁵ copies/mg or more were detected at 14 days after injection (10⁷ copies/fish) in brain tissues of 4 species except for crucian carp and common carp among the 6 species of juvenile fish used. In addition, the histopathological features of weak but distinct vacuole formation were also found in the brain of these infected fish, but no difference was found between the two reassortants KS/RGNNV-KG and RG/KSNNV-CM.

• 한국균학회지
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• 제41권3호
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• pp.200-204
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• 2013

일부 자낭균류는 Dihydroxynaphthalene(DHN) 멜라닌을 가지고 있다. 본 연구는 배에 검은별무늬병을 일으키는 V. nashicola 균이 어떠한 형태의 멜라닌을 가지고 있는지 확인하고자 DHN 멜라닌 합성유전자중의 하나인 scytalone dehydratase(SD) 유전자의 부분 염기서열을 국내 여러 지역과 일본에서 분리된 11개 균주로부터 분석하였다. PCR 방법을 사용하여 429 bp 크기의 반응산물을 11개 균주 모두로부터 증폭하였고 염기서열을 분석하였다. 증폭된 PCR 산물은 GenBank database에 비교 탐색한 결과 SD 유전자로 판정되었다. 분석된 11개 균주의 SD 유전자에는 모두 1개의 인트론과 122개 아미노산을 코딩하는 2개의 엑손이 존재하였다. 이들 11개 SD 유전자 간에 염기서열은 100% 상동성을 보였다. 결정된 V. nashicola SD 유전자의 아미노산 서열의 유사도는 다른 곰팡이와 비교할 때 69~73%로 수준이었다. 본 연구 결과는 V. nashicola 균이 DHN 멜라닌 생합성 단계를 운영하고 있음을 입증하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권6호
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• pp.891-895
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• 2019

Objective: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the ${\alpha}$-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). Methods: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). Results: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources ($0.25ng/{\mu}L$). Conclusion: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

• 대한본초학회지
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• 제37권4호
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• pp.9-16
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• 2022

Objectives : In the Korean Pharmacopoeia 12th edition (KP 12) and the Korean Herbal Pharmacopoeia (KHP), two authentic herbal medicines are described, namely Bang-gi (Cheong-pung-deung) and Mok-bang-gi, respectively. In China, Bun-bang-gi is also used as herbal medicine. This study was conducted to develop a molecular authentication tool for distinguishing the three herbal medicine used as Bang-gi, which are Sinomeni Caulis et Rhizoma (Rhizome of Sinomenium acutum), Stephaniae Tetrandrae Radix (Root of Stephania terandra), and Cocculi Radix (Root of Cocculus trilobus). Methods : Twelve samples of three species (four samples of S. acutum, five samples of S. tetrandra, and three samples of C. trilobus) were collected from different habitats. The sequences of internal transcribed spacer (ITS) regions were obtained and comparatively analyzed to design the species-specific sequence characterized amplified region (SCAR) primers. The specificity of each pair of SCAR primers that amplified species-specific amplicon was evaluated for establishing the singleplex and multiplex PCR assay tools. Results : The singleplex SCAR markers show discriminability in C. acutum, S. tetrandra, and C. trilobus. These SCAR markers were also efficiently authenticated three species in the multiplex SCAR amplification using single PCR reaction. Furthermore, these PCR assay methods were applicable to authenticate dried herbal medicines distributed in the markets. Conclusions : The SCAR markers and PCR assay tools help discriminate the three herbal medicines used as Bang-gi at the species levels and provide a reliable genetic method to prevent the inauthentic distribution of these herbal medicines.

• Mycobiology
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• 제40권2호
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• pp.138-141
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• 2012

We measured physiological functionalities, including antihypertensive angiotensin I-converting enzyme inhibitory activity and immun-stimulating ${\beta}$-glucan content for sixty kinds of Makgeolli that is commercially available from the market. As a result, we selected R-12 commercial raw Makgeolli, with a high content of immuno-stimulating ${\beta}$-glucan, and R-14 commercial raw Makgeolli, exhibiting high antihypertensive activity. Due to the similarities in their overall physicochemical properties and raw materials used for fermentation, we compared the microbial flora in order to investigate the reason for the differences in their functionalities. Nested PCR and denaturing gradient gel electrophoresis for yeasts and bacteria were performed for analysis of microbial diversity of two different kinds of Makgeolli (i.e., R-12, R-14), which showed immuno-stimulating ${\beta}$-glucan content and exhibited a very high level of antihypertensive activity, respectively. Analysis of the 18S rDNA amplicon revealed a major presence of the yeast strain Pichia burtonii in every Makgeolli sample. Analysis of the 16S rDNA amplicon revealed a predominance of lactic acid bacteria, and the most frequent lactic acid bacteria were Lactobacillus ingluviei, L. fermentum, and L. harbinensis, and Lactobacillus sp. Among these, L. harbinensis was detected only in R-12 and L. ingluviei was found only in R-14. Different functionalities from the individual commercially available Makgeolli may be attributed to actions of different microbial flora during fermentation.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.856-867
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• 2017

Supplement of dietary fibers (DF) is regarded as one of the most effective way to prevent and relieve chronic diseases caused by long-term intake of a high-fat diet in the current society. The health benefits of soluble dietary fibers (SDF) have been widely researched and applied, whereas the insoluble dietary fibers (IDF), which represent a higher proportion in plant food, were mistakenly thought to have effects only in fecal bulking. In this article, we proved the anti-obesity and glucose homeostasis improvement effects of IDF from pear pomace at first, and then the mechanisms responsible for these effects were analyzed. The preliminary study by real-time PCR and ELISA showed that this kind of IDF caused more changes in the gut microbiota compared with in satiety hormone or in hepatic metabolism. Further analysis of the gut microbiota by high-throughput amplicon sequencing showed IDF from pear pomace obviously improved the structure of the gut microbiota. Specifically, it promoted the growth of Bacteroidetes and inhibited the growth of Firmicutes. These results are coincident with previous hypothesis that the ratio of Bacteroidetes/Firmicutes is negatively related with obesity. In conclusion, our results demonstrated IDF from pear pomace could prevent high-fat diet-induced obesity in rats mainly by improving the structure of the gut microbiota.

• Asian-Australasian Journal of Animal Sciences
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• 제24권7호
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• pp.898-904
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• 2011

The PCR- restriction fragment length polymorphism (RFLP) in and around TM4 of SLC11A1 gene and its association with the incidences of brucellosis in Hariana breed (Bos indicus) and Holstein Friesian crossbred (Bos indicus${\times}$Bos taurus) cattle was examined. A fragment of 954 bp encoding the TM4 was amplified, and RFLP was identified by digestion of the amplicon independently with AluI and TaqI. The amplicon (GenBank Acc. No. AY338470 and AY338471) comprised of a part of exon V (<59 bp) and VII (62>), and entire intron 5 (423 bp), exon VI (71 bp) and intron 6 (339 bp). Digestion with AluI revealed the presence of two alleles viz, A (281, 255, 79 and 51 bp) and B (541, 255, 79 and 51 bp). The frequency of A allele was estimated as 0.80 and 0.73 in Hariana and crossbred cattle, respectively. Due to presence of a polymorphic TaqI site at intron 5, two alleles: T (552 and 402 bp) and Q (231, 321 and 402 bp) were identified. The frequency of T allele was estimated as 0.96 and 0.97, respectively. For association study, on the basis of serological tests and history of abortion, the animals were grouped into "affected" and "non-affected". However, no association could be established with the observed RFLPs.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7935-7941
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• 2015

The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage ($>25{\times}$) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.