• 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.

• 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.

• Environmental Engineering Research
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• v.20 no.1
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• pp.51-57
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• 2015

As expected, the expression of denitrifying genes in a Typha wetland (relatively stagnant compared to other ponds), showing higher nitrogen removal efficiency in summer, was affected by temperature. The abundance and gene transcripts of nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ) genes in seasonal sediment samples taken from the Acorus and Typha ponds of free surface flow constructed wetlands were investigated using quantitative polymerase chain reaction (Q-PCR) and quantitative reverse transcription PCR (Q-RT-PCR). Denitrifying gene copy numbers ($10^5-10^8$ genes $g^{-1}$ sediment) were found to be higher than transcript numbers-($10^3-10^7$ transcripts $g^{-1}$ sediment) of the Acorus and Typha ponds, in both seasons. Transcript numbers of the four functional genes were significantly higher for Typha sediments, in the warm than in the cold season, potentially indicating greater bacterial activity, during the relatively warm season than the cold season. In contrast, copy numbers and expression of denitrifying genes of Acorus did not provide a strong correlation between the different seasons.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.1-6
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• 2021

Since the outbreak of coronavirus disease 2019 (COVID-2019), the infection has spread worldwide due to the highly contagious nature of severe acute syndrome coronavirus (SARS-CoV-2). To manage SARS-CoV-2, the development of diagnostic assays that can quickly and accurately identify the disease in patients is necessary. Currently, nucleic acid-based testing and serology-based testing are two widely used approaches. Of these, nucleic acid-based testing with quantitative reverse transcription-PCR (RT-qPCR) using nasopharyngeal (NP) and/or oropharyngeal (OP) swabs is considered to be the gold standard. Recently, the use of saliva samples has been considered as an alternative method of sample collection. Compared to the NP and OP swab methods, saliva specimens have several advantages. Saliva specimens are easier to collect. Self-collection of saliva specimens can reduce the risk of infection to healthcare providers and reduce sample collection time and cost. Until recently, the sensitivity and accuracy of the data obtained using saliva specimens for SARS-CoV-2 detection was controversial. However, recent clinical research has found that sensitive and reliable data can be obtained from saliva specimens using RT-qPCR, with approximately 81% to 95% correspondence with the data obtained from NP and OP swabs. These data suggest that self-collected saliva is an alternative option for the diagnosis of COVID-19.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• BMB Reports
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• v.30 no.3
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• pp.234-236
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• 1997

Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.816-824
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• 2017

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

• 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.

• Journal of fish pathology
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• v.19 no.3
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• pp.189-195
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• 2006

A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.