• Genomics & Informatics
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• 제21권2호
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• 한국콘텐츠학회:학술대회논문집
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• 한국콘텐츠학회 2018년도 춘계 종합학술대회 논문집
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• pp.561-562
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• 2018

디지털 중합효소연쇄반응(Digital PCR)은 3세대 PCR로 명명하며, 1세대인 일반 PCR과 2세대인 정량 PCR(Real-time PCR)의 단점을 보완하여 개발된 방법이다. Digital PCR System은 소량의 PCR 반응을 10만개 이상의 반응통(wall)에 적재하는 방식의 나노유체칩에서 쪼개어 증폭시킨 후, target DNA를 계수한다. Target DNA의 증폭 여부에 따라 positive(1)와 negative(0)로 digital signal처럼 받아들여 계수하고, 포아송 분포를 통해 target DNA의 copy를 계산해 최종적으로 샘플 microlitr당 Copy수로 결과 값을 확인할 수 있다. 본 연구에서는 종(種)이 다른 동물의 조직이 서로 섞여있을 때 사람의 조직을 탐색하는 방법으로 유전자 증폭을 할 경우, digital PCR의 유효성에 대해 증명하였다.

• Genomics & Informatics
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• 제21권4호
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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.

• Genomics & Informatics
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• 제19권3호
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• 식물병연구
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• 제27권3호
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• pp.120-127
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• 2021

식물 바이러스는 작물 수확량에 상당한 손실을 일으키고 작물 생산을 지속적으로 위협하여 세계 식량 안보에 심각한 위협이 된다. 그 중 tomato spotted wilt virus (TSWV)는 주로 원예작물을 감염시키는 가장 위협적인 식물 바이러스로 넓은 기주 범위를 가진다. Reverse-transcription quantitative real-time PCR (RT-qPCR)은 TSWV의 민감한 검출을 위해 널리 사용되고 있지만 표준화의 어려움으로 인해 유용성이 감소한다. 따라서 본 연구에서는 TSWV 검출을 위해 민감하고 정확한 reverse transcription droplet digital polymerase chain reaction (RT-ddPCR)을 확립하였다. TSWV 검출에 대한 RT-qPCR 및 RT-ddPCR의 민감도를 비교하였고, TSWV에 대한 RT-ddPCR의 특이성 분석은 고추에서 주로 발생하는 바이러스 및 음성 대조군에서 특이성을 확인한 결과 증폭되지 않았다. RT-ddPCR 및 RTqPCR에 의해 측정된 TSWV의 선형회귀곡선은 모두 높은 선형성을 나타냈지만, RT-ddPCR 분석이 10배 이상 더 민감하고 더 낮은 TSWV의 copy 수를 검출할 수 있었다. 종합적으로, 우리의 연구 결과는 RT-ddPCR이 TSWV 검출에 대해 높은 민감도와 특이성을 제공하고 낮은 농도의 현장 시료에서 TSWV 검출하는 데 적합하다는 것을 보여준다.

• Genomics & Informatics
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• 제18권1호
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• pp.4.1-4.6
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• 2020

Transposable elements (TEs) constitute approximately half of Bovine genome. They can be a powerful species-specific marker without regression mutations by the structure variation (SV) at the time of genomic evolution. In a previous study, we identified the Hanwoo-specific SV that was generated by a TE-association deletion event using traditional PCR method and Sanger sequencing validation. It could be used as a molecular marker to distinguish different cattle breeds (i.e., Hanwoo vs. Holstein). However, PCR is defective with various final copy quantifications from every sample. Thus, we applied to the droplet digital PCR (ddPCR) platform for accurate quantitative detection of the Hanwoo-specific SV. Although samples have low allele frequency variation within Hanwoo population, ddPCR could perform high sensitive detection with absolute quantification. We aimed to use ddPCR for more accurate quantification than PCR. We suggest that the ddPCR platform is applicable for the quantitative evaluation of molecular markers.

• 미생물학회지
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• 제43권3호
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• pp.232-235
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• 2007

해양 및 토양에서의 암모니아 산화는 세균에 비해 Crenarchaeota 그룹의 archaea에 의해 우세하게 일어나고 있음이 알려졌다. 서해 갯벌에서, 배양에 의존하지 알고, 특정 암모니아 산화유전자(amoA)를 가진 archaea을 동정하고자 디지털 PCR법을 응용한 nested PCR법을 개발하였다. amoA와 16S rRNA유전자가 동시에 증폭된 샘플의 분석결과, 16S rRNA유전자에 비해 amoA 유전자의 다양성 이 높았으며, I.1a 그룹의 crenarchaea가 I.1b 그룹의 crenarchaea보다 갯벌지역에서 암모니아 산화에 우점적으로 기여하고 있음을 알 수 있었다. 본 연구에서 시도된, 디지털 PCR과 multiplex-nested PCR을 접목한 접근법을 이용하면 특정 기능유전자를 가진 미생물을 환경에서 검증하는데 응용할 수 있을 것이다.

• ALGAE
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• 제39권1호
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• pp.51-56
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• 2024

The use of droplet digital polymerase chain reaction (ddPCR) has greatly improved the quantification of harmful protists, outperforming traditional methods like quantitative PCR. Notably, ddPCR provides enhanced consistency and reproducibility at it resists PCR inhibitors commonly found in environmental DNA samples. This study aimed to determine the most effective filter material for ddPCR protocols by assessing the reproducibility of species-specific gene copy numbers and filtration time across six filter types: cellulose acetate (CA), mixed cellulose ester (MCE), nylon (NY), polycarbonate (PC), polyethersulfone (PES), and polyvinylidene fluoride (PVDF). The study used two species of Chattonella marina complexes as a case study. Filtration rates were slower for NY, PC, and PVDF filters. Moreover, MCE, NY, PES, and PVDF yielded lower DNA amounts than other filters. Importantly, the CA filter exhibited the lowest variance (38-39%) and the highest determination coefficients (R² = 0.92-0.96), indicating superior performance. These findings suggest that the CA filter is the most suitable for ddPCR quantification of marine protists, offering quick filtration and reliable reproducibility.

• 한국식품과학회지
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• 제48권5호
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• pp.454-460
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• 2016

본 논문에서는 E. coli O157:H7, S. Typhimurium, S. aureus와 B. cereus에 대한 ddPCR의 검출 효율과 검출한계를 측정하였으며 동시 검출을 위한 multiplex 검출 가능성을 타진하였다. ddPCR은 PCR mix를 포함한 시료를 15,000-20,000개의 droplet으로 분할하여 PCR 하는 방법으로 droplet reader를 이용하여 droplet의 형광 신호를 계수하였다. 식중독 세균의 표적 DNA를 검출하기 위해 2가지 색의 형광 probe (TaqMan)를 제작하였다. ddPCR은 표적 유전자의 형광 신호를 $100fg/{\mu}L$부터 $10ng/{\mu}L$까지의 DNA를 검출 할 수 있었다. 이후에 두 종류의 식중독 세균에서 프라이머 농도를 달리하여 표적 DNA 증폭 크기의 분포가 서로 다르게 구별할 수 있음을 확인하여 multiplex PCR의 가능성이 있음을 알 수 있었다. ddPCR은 비교적 낮은 검출한계를 가지기 때문에 식품에 적은 농도로 존재하는 식중독 세균의 검출에 활용이 가능할 것으로 생각되었다. 또한 식품의 전처리 조건 확립과 반응조건 확립을 통하여 향후 복잡한 matrix effect를 가지는 식품에서 극 미량의 균 검출도 가능할 것으로 생각되었다.

• The Plant Pathology Journal
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• 제38권4호
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• pp.417-422
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• 2022

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.