• 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.

• 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.

• ALGAE
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• v.32 no.3
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• pp.189-197
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• 2017

To quantify the abundance of the harmful dinoflagellate Cochlodinium polykrikoides in natural seawaters, we developed the innovative procedure using a droplet digital PCR (ddPCR) with C. polykrikoides-specific primers targeting the internal transcription sequence (ITS). The abundance of C. polykrikoides was estimated by the specific copy number of target ITS DNA segments per cell in cultures and natural water samples. The copy number per C. polykrikoides cell as acquired by ddPCR was $157{\pm}16$, which was evaluated against known cell numbers through a simplified protocol preparing DNAs. The abundances of C. polykrikoides in the waters of different locations estimated by ddPCR agreed with the number of cells visually counted under a microscope. This protocol was used to measure the abundance of C. polykrikoides close to and further off the southern coast of Korea in August of 2016 and 2017. The practical application showed that this method can reduce time for analysis and increase accuracy.

• Journal of Mushroom
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• v.2 no.3
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• pp.145-148
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• 2004

This study was carried out to screen the fruiting body formation-specific genes from the medicinal mushroom Cordyceps militaris. A cDNA synthesized using total RNA from 4 stages of mushroom development, mycelium, primordium, immature fruiting body and mature fruiting body. Differential expression gene screening was performed by DD-PCR(Differential Display Arbitrary Primer PCR) with cDNA, we sequenced partial 6 genes using pGEM cloning vector. The DNA Sequence of the six DD-PCR products derived from differentially expressed genes was compared to that in the GenBank database by using the NCBI BLAST search to identify similarities to known sequences. Sequence analysis showed that six of DD-PCR products have unknown sequence.

• 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.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2006.11a
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• pp.359-361
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• 2006

As circuits become increasingly complex and devices sizes shrinks, the demands placed on global planarization of higher level. Chemical Mechanical Polishing (CMP) is an indispensable manufacturing process used to achieve global planarity. In the CMP process, Diamond Disk (DD) plays an important role in the maintenance of removal rate. According to studies, the cause of removal rate decrease in the early or end stage of diamond disk lifetime comes from pad surface change. We also presented pad cutting rate (PCR) as a useful cutting ability index of DD and studied PCR trend about variable parameters that including size, hardness, shape of DD and RPM, pressure of conditioner It has been shown that PCR control ability of pressure and shape is superior to RPM and size. High pressure leads to a decrease of cell open ratio of pad surface because polyurethane of pad is destroyed by pressure. So low pressure high RPM condition is a proper removal rate sustain. By examining correlations between RPM and pressure of conditioner, it has been shown that PCR safe zoneto satisfy proper removal rate has the range 0.06mm/hr to 0.12mm/hr.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.358-367
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• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

• 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.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.167-173
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• 2012

This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as ${\beta}$-glycerophosphate (${\beta}$-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.