• Asian-Australasian Journal of Animal Sciences
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• 제19권10호
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• pp.1503-1507
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• 2006

Goats' milk adulteration with cows' milk is becoming a big problem. In the past, the urea-polyacrylamide gel electrophoresis assay with different motility of ${\alpha}S1$-casein has been applied for the identification of cows' milk adulteration. The detection sensitivity is 1.0%. The aim of this study was to develop a faster and more sensitive method to detect cows' milk which may be present in adulterated goats' milk and goats' milk powder. The published primer was targeted at highly conserved regions in bovine mitochondrial DNA (a 271 bp amplicon). This amplicon was cloned and sequenced to further confirm bovine specific sequence. The chelex-100 was used to separate bovine somatic cells from goats' milk or goats' milk powder samples. Random sampling of different brands of goats' milk powder and tablets from various regions of Taiwan showed the adulterated rate was 20 out of 80 (25%) in goats' milk powders and 12 out of 24 (50%) in goats' milk tablets. With this system, as low as 0.1% cows' milk or cows' milk powder in goat milk or goat milk powder could be identified. This chelex DNA isolation approach provides a fast, highly reproducible and sensitive method for detecting the adulteration of goats' milk products.

• 식물병연구
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• 제21권3호
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• pp.180-185
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• 2015

In the present work, a pair of primers specific to Tomato yellow leaf curl virus (TYLCV) was designed to allow specific amplification of DNA fragments from any TYLCV isolates using an extensive alignment of the complete genome sequences of TYLCV isolates deposited in the GenBank database. A pair of primers which allows the specific amplification of tomato ${\beta}$-tubulin gene was also analyzed as an internal PCR control. A duplex PCR method with the developed primer sets showed that TYLCV could be directly detected from the leaf crude sap of infected tomato plants. In addition, our developed duplex PCR method could determine PCR errors for TYLCV diagnosis, suggesting that this duplex PCR method with the primer sets is a good tool for specific and sensitive TYLCV diagnosis. The developed duplex PCR method was further verified from tomato samples collected from some farms in Korea, suggesting that this developed PCR method is a simple and reliable tool for rapid and large-scale TYLCV detections in tomato plants.

• 한국콘텐츠학회논문지
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• 제22권10호
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• pp.733-741
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• 2022

해양범죄에서 발견되는 다양한 증거물 중 지문과 DNA(Deoxyribo nucleic acid)는 용의자를 특정할 수 있다는 점에서 매우 중요하다. 본 연구에서는 실제 해양환경에서 증거물로 많이 발견되는 비다공성 재질 5종(플라스틱, 스테인리스, 유리, 세라믹, FRP(Fiber reinforced plastic))을 선정하여 자연 및 혈액지문을 유류한 후 동해 해경서 전용부두에서 약 7일간 침지하였다. 그 후 CA(Cyanoacrylate) 훈증법과 4가지 분말법(Swedish black powder, Concentrated black powder, Supranano red powder, Dazzle orange powder)을 이용하여 지문 현출 후 DNA 추출, 정량, STR(Short tandem repeat) 프로필을 분석하였다. 지문현출방법 중 Supranano red powder를 적용하였을 때 DNA 농도가 상대적으로 높은 양이 나타났으며 STR 프로필 분석을 실시한 결과 평균 16.8~9개의 유전자좌위를 확보할 수 있었고, 유리 및 세라믹 재질에서는 20개 모두 확인할 수 있었다. 연구 결과 약 7일 동안 침지된 가상증거물에 지문 현출법을 적용 후 DNA를 추출 및 정량하여 STR 프로필을 확보할 수 있었으며, VMD(Vacuum metal deposition), SPR(Small particle reagent) 등 다양한 지문현출방법을 적용한 뒤 DNA를 분석하여 STR 프로필을 확보할 수 있는 추가적인 연구가 필요할 것으로 판단된다.

• 보존과학연구
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• 통권23호
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• pp.59-75
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• 2002

In this study human bones and teeth, excavated from the Myeongam-risite in Asan, Chungcheongnam-do Province, have been analysed by nuclear DNA typing and mitochondrial DNA sequencing methods. Twenty-one samples of long bones and twenty-seven samples of teeth from twenty-one individuals were collected and analysed. Among these thirteenteeth were successfully subjected to nuclear DNA extraction, quantification, and PCR(Polymerase Chain Reaction) amplification. Silver STR III (D16S539, D7S820, D13S317) multiplex PCR method was used in this study for a short tandem repeat (STR) analysis. Mitochondrial DNAs of tooth samples were also amplified and sequenced by a DNA sequencer. These analyses show that a sample from Burial no. 29 and one from Burial no. 38(right) possessed the same maternal inheritance. This may suggest that the Myeongam-ri cemetery was used by a kin group for a relatively long period of time.

• Archives of Pharmacal Research
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• 제19권6호
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• pp.486-489
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• 1996

A simple, rapid, specific and sensitive method for the detection of serum hepatitis C virus (HCV) RNA using the reverse transcription-polymerase chain reaction (RT-PCR) technique without conventional RNA extraction was developed. HCV template RNA from serum was obtained by boiling the serum at $95^{\circ}C$ for 2 min, cooling rapidly in ice and removing the proteins by cetrifugation. RT-PCR amplifications including the reverse transcription and first PCR amplification were performed in one vessel containing both of reverse transcriptase and Taq DNA polymerase. The detection of HCV RNA from $10^{-3}{\mu}l$. serum was possible with this method. The suitability of this method for clinical analysis was evaluated by assaying HCV RNA in 225 patient samples including anti-HCV antibody negatives (13 samples) and positives (212 samples) by enzyme-linked immunosorbent assay test (ELISA). Detections of HCV RNA with this method were in 4 of 13 anti-HCV antibody negative samples (30.8%) and 95 of 212 positive samples (44.8%). The present method can be completed in 1 hr and has a wide range of application for the clinical utilities to determine the viral RNAS.

• 한국수산과학회지
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• 제40권6호
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• pp.356-361
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• 2007

Recently, the development of various culture-independent identification techniques for environmental microbes has greatly enhanced our knowledge of microbial diversity. In particular, denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments, generated using the polymerase chain reaction (PCR) is frequently used to examine the diversity of environmental bacterial populations. This method consists of direct extraction of the environmental DNA, amplification of the 200-600 bp 16S rDNA fragments with universal primers, and separation of the fragments according to their melting point on a denaturing gradient gel. In this study, we investigated the seaside microbial community in coastal areas of Busan, Korea, using culture-independent techniques. First, marine genomic DNA was extracted from seawater samples collected at Songjeong, Gwangahn, and Songdo Beaches. Then, PCR was used to amplify the bacterial 16S rDNA using universal primers, and DGGE was used to separate the amplified 500 bp 16S rDNA fragments. Finally, the tested 16S rDNA genes were further analyzed by sequencing. Based on these experiments, we found that DGGE analysis clearly showed variation among the regional groups. It can be used to monitor rapid changes in the bacterial diversity of various environments. In addition, the sequence analysis indicated the existence of many unculturable bacteria, in addition to Arcobacter, Pseudoaltermonas, and Vibrio species.

• 미생물학회지
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• 제34권3호
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• pp.132-136
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• 1998

자연환경의 미생물군집을 분자생태학적 방법으로 분석하기 위해서는 오염되지 않은 순수한 핵산의 분리가 필요하다. 해양퇴적물에서 핵산을 추출할 때 같이 추출되는 부식물질(humic substances)은 핵산중폭반응을 저해하기 때문에, 이를 제거하기 위한 네 가지의 방법, 아가로즈젤 전기영동후 용출, Sephadex G-75 젤, Hydroxyapatite 젤, 그리고 PVPP(poluvinylpolypyrrolidone) microspin column 방법에 대해 그 효율성을 비교하였다. 조산된 방법 중에서 PVPP microspin column을 이용하면 건조 퇴적물 1g당 $4.8{\mu}g$의 순수한 DNA를 신속하고 간편하게 얻을 수 있었고, 이 방법으로 분리된 DNA를 PCR의 주형으로 사용한 결과 16S rRNA 유전자의 거의 전 범위에 해당하는 1.5 kb 크기의 PCR 산물을 얻을 수 있었다.

• Journal of Plant Biotechnology
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• 제47권1호
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• pp.15-25
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• 2020

Allium L. is one of the largest genera of the Amaryllidaceae family, with more than 920 species including many economically important species used as vegetables, spices, medicines, or ornamental plants. Currently, DNA barcoding tools are being successfully used for the molecular taxonomy of Allium. A total of 46 Allium species were collected from their native areas, and DNA was extracted using the IBRC DNA extraction kit. We used specific primers to PCR amplify matK. DNA sequences were edited and aligned for homology, and a phylogenetic tree was constructed using the neighbor-joining method. The results show thymine (38.5%) was the most frequent and guanine (13.9%) the least frequent nucleotide. The matK regions of the populations were quite highly conserved, and the amount of C and CT was calculated at 0.162 and 0.26, respectively. Analysis of the nucleotide substitution showed C-T (26.22%) and A-G (8.08%) to have the highest and lowest percent, respectively. The natural selection process dN/dS was 1.16, and the naturality test results were -1.5 for Tajima's D and -1.19 for Fu's Fs. The NJ dendrogram generated three distinct clades: the first contained Allium austroiranicum and A. ampeloprasum; the second contained A. iranshahrii, A. bisotunense, and A. cf assadi; and the third contained A. rubellum and other species. In this study, we tested the utility of the matK region as a DNA barcode for discriminating Allium. species.

• 대한한의학회지
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• 제23권2호
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• pp.57-69
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• 2002

Objective: The aim of this study is to investigate the effect of Injin butanol fractions with Thin Layer Chromatography on Fas-mediated Apoptosis. Method: Injin-butanol fraction separated by TLC. MIT assay, cell cycle analysis, Caspase-3 protease assay, DNA fragmentation assay and quantitative RT-PCR were performed to evaluate the effects of TLC extraction of lnjin-butanol fraction on cell viability, cell cycle progression and apoptosis. Results: Scopoletin, luteolin, apigenin and unknown powder was isolated by TLC. Fas-mediated apoptosis analysis shows that scopoletin has inhibiting function on apoptosis. Caspase- 3 protease assay analysis shows that scopoletin inhibits activity of caspase-3. Quantitative RT-PCR analysis shows that no activity on caspase-3, but apoptosis inhibition cytokine -Bcl-2- is activated, and apoptosis activating cytokine -Bax- is unactivated. Conclusion: These results show that each fraction of Injin-butanol TLC extraction, especially scopoletin, acts as a protective function on liver cell viability, and inhibitory function on apoptosis. (J Korean Oriental Moo 2002;23(2):57-69)

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1401-1409
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• 2015

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10⁰ fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.