• Genomics & Informatics
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• v.21 no.2
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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.

• Proceedings of the Korea Contents Association Conference
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• 2018.05a
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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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• v.21 no.4
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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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• v.19 no.3
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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.

• Research in Plant Disease
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• v.27 no.3
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• pp.120-127
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• 2021

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

• Genomics & Informatics
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• v.18 no.1
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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.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.232-235
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• 2007

Mesophilic Crenarchaeota have been known to be predominant among ammonia-oxidizing microorganisms in terrestrial and marine environments. In this study, we determined the archaeal phylotypes carrying specific amoA by combining digital PCR and multiplex-nested PCR. Analysis of samples in which amoA and 16S rRNA gene were amplified showed that amoA gene diversity was relatively higher than that of 16S rRNA gene. Nitrifying archaeal group I.1a was dominant over I.1b group of crenarchaota and euryarchaeota. This approach could be applied for interrelating a functional gene to a specific phylotype in natural environments.

• ALGAE
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• v.39 no.1
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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.

• Korean Journal of Food Science and Technology
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• v.48 no.5
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• pp.454-460
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• 2016

In this study, we investigated the possibility of Droplet digital PCR (ddPCR) for detection of foodborne pathogens. ddPCR combines partitioning of PCR reactions into several thousands or millions of individual droplets in a water-oil emulsion, and counting of positive PCR reaction using flow cytometry. Four species of foodborne pathogens, Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli O157:H7, were used to quantify the target sequence with each of the designed primers and double stranded DNA-binding Evagreen dye. All tested foodborne pathogens showed a detection limit ranging from $100fg/{\mu}L$ to $10ng/{\mu}L$. It was concluded that ddPCR could be used to detect very low concentrations of foodborne pathogens from complex food matrices. For multi-detection of target pathogens, we also tested the samples using multiplex ddPCR and obtained successful results.

• The Plant Pathology Journal
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• v.38 no.4
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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.