• 대한수의학회지
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• 제57권1호
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

• 한국미생물·생명공학회지
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• 제47권2호
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• pp.317-322
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• 2019

Gastroenteritis with diarrhea is one of the most infectious diseases in the world following respiratory infections. Notably, diarrhea-causing viruses (DVs) cause more than 70% of such cases. In this study, 3,065 stool specimens from patients with diarrhea (median age, 1.1 years; range, 0.0-91.1 years), who were admitted to the DanKook University Hospital, were examined using multiplex reverse transcription PCR (mRT-PCR). The target viruses were astrovirus (AstV), enteric adenovirus (EAdV), group A rotavirus (RotV), norovirus GI (NoV-GI), and norovirus GII (NoV-GII). The mRT-PCR results were analyzed based on various factors such as seasonality, age, presence of co-infection, and analyzed trends. The detection rate of the DVs during the study period was found to be 30.8% (n = 943/3,065). When the detection rate was analyzed monthly, the DV detection rate was found to be highest between December to January. Of the detected DVs, NoV-GII was the most common, accounting for 45.5% of the detected viruses (n = 446/980). Notably, 86.5% (n = 848/980) of the pathogens were detected in individuals who were less than 5 years of age. During the study period, NoV-GII and RotV showed alternating trends. In addition, both the number and rate of co-infections increased.

• The Plant Pathology Journal
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• 제34권6호
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• 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.

• 식물병연구
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• 제28권2호
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• pp.92-97
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• 2022

Apple stem grooving virus (ASGV) is a high-risk viral pathogen that infects many types of fruit trees, especially pear and apple, and causes serious economic losses across the globe. Thus, rapid and reliable detection assay is needed to identify ASGV infection and prevent its spread. A reliable reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed, optimize, and evaluated for the coding region of coat protein of ASGV in pear leaf. The developed RT-LAMP facilitated the simple screening of ASGV using visible fluorescence and electrophoresis. The optimized reaction conditions for the RT-LAMP were 63℃ for 50 min, and the results showed high specificity and 100-fold greater sensitivity than the reverse transcription polymerase chain reaction. In addition, the reliability of the RT-LAMP was validated using field-collected pear leaves. Furthermore, the potential application of paper-based RNA isolation, combined with RT-LAMP, was also evaluated for detecting ASGV from field-collected samples. These assays could be widely applied to ASGV detection in field conditions and to virus-free certification programs.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1997년도 한국생물과학협회 학술발표대회
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• pp.78-78
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• 1997

No Abstract, See Full Text

• 한국식물학회:학술대회논문집
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• 한국식물학회 1998년도 The 12th Symposium on Plant Biotechnology Vol.12
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• pp.116-116
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• 1998

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• The Plant Pathology Journal
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• 제38권6호
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• pp.665-672
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• 2022

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39℃) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

• Journal of Photoscience
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• 제7권2호
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• pp.73-78
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• 2000

To analyze molecular traits determining pigmentation between Pharbitis nill violet and white, Amplified Fragment Length Polymorphism(AFLP) and Differential Display Reverse Transcription(DDRT) experiments were carried out with either genomic DNAs or total RNAs isolated from both plants. Results of AFLP experiment in combination of 8 EcoRⅠ primers with 6 MseⅠ primers showed 41 violet-and 60 white-specific DNA bands. In the subsequent experiment, 22 violet-and 22 white-specific DNA fragments were amplified by PCR with DNAs eluted. The sizes of the fragments range from 200 to 600bp. DDRT using total RNA produced 19 violet-and 17 white-specific cDNA fragments, ranging from 200 to 600bp. The fragments obtained by both AFLP and DDRT had been cloned into pGEM T-easy vector, amplified and subjected to the nucleotide sequence analyses. As a result of Blast sequence analysis, most of them sequenced up to date showed no similarity to any Known gene, while few has similarity to known animal or plant genes. An AFLP clone V6, for example, has a strong sequence similarity to the human transcription factor LZIP-alpha mRNA and a DDRT clone W19 to Solanum tuberosum 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA.