• Horticultural Science & Technology
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• v.18 no.4
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• pp.513-517
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• 2000

This experiment was carried out to detect the cymbidium mosaic virus (CymMV) and the odontoglossum ringspot virus (ORSV) in the seed-derived plantlets of Phalaenopsis imported from Taiwan by one-step reverse transcription-polymerase chain reaction (RT-PCR). Simple and rapid crude plant extracts for RT-PCR were prepared. The reverse transcription step was performed at $42^{\circ}C$ for 45 min and the following thermal cycling scheme was used for 36 reaction cycles: template predenaturation at $96^{\circ}C$ for 2 min, template denaturation at $96^{\circ}C$ for 30 s, primer annealing at $60^{\circ}C$ for 30 s, and DNA synthesis at $72^{\circ}C$ for 1 min. Of the 40 seed-derived plantlets of Phalaenopsis imported from Taiwan, all of them were infected with CymMV, but ORSV was not detected.

• Korean Journal of Veterinary Service
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• v.46 no.4
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• pp.269-281
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• 2023

In this study, a new triplex real-time quantitative reverse transcription polymerase chain reaction (tqRT-PCR) assay was developed for the rapid and differential detection of three feline viral pathogens including feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and influenza A virus (IAV) in a single reaction. The assay specifically amplified three targeted viral genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 1%. Based on the diagnostic results of the assay using 120 clinical samples obtained from cats with feline respiratory disease complex (FRDC)-suspected signs, the prevalence of FCV, FHV-1, or IAV was 43.3%, 22.5%, or 0%, respectively, indicating that the diagnostic sensitivity was comparable or superior to those of previously reported monoplex qRT-PCR/qPCR assays. The dual infection rate for FCV and FHV-1 was 8.3%. These results indicate that FCV and FHV-1 are widespread and that co-infection with FCV and FHV-1 frequently occur in the Korean cat population. The developed tqRT-PCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens, and the prevalence data for three feline viruses obtained in this study will contribute to expanding knowledge about the epidemiology of FRDC in the current Korean cat population.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.378-384
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• 2011

Tomato yellow leaf curl virus (TYLCV), a member of genus Begomovirus, was isolated in Korea in 2008. We sequenced and analyzed the DNA-A of 51 TYLCV isolates from Korea, and 13 of the TYLCV isolates were selected as type representatives of TYLCV from six Korean provinces. The 13 TYLCV isolates were classified into Korea Group 1 (KG1, nine isolates) and Korea Group 2 (KG2, four isolates) based on the results of phylogenetic analysis and genome size (2774 and 2781 nucleotides, respectively). A recombination detection program 3 (RDP3) revealed two recombinations between the TYLCV Korea isolates and other TYLCV isolates [Thailand (AF206674), Iran (AJ132711), and Israel (X76319)]. TYLCV Jeju isolate was characterized by two recombination events (E1 and E2) caused by the presence of E1 in ORF V1 and C3, which may seem to be the mutations of the high nucleotide transversion and transition rate. Collectively, our results suggest that the occurrence of nucleotide transversions and transitions in TYLCV DNA-A might have induced novel recombination events within the TYLCV Korea isolates.

• The Plant Pathology Journal
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• v.19 no.5
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• pp.260-265
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• 2003

Gummy stem blight caused by Didymella bryoniae occurs exclusively in cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. In this study, cultural conditions for the mass-production of pycnidiospore by Metal Halide (MH) lamp irradiation were maximized. The mycelia were cultured at $26^{\circ}C$ on PDA for 2 days under dark condition, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$. Results show that a great number of pycnidia were simultaneously formed. The pycnidiospores produced were then used as immunogen. Fusions of myeloma cell (v-653) with splenocytes from immunized mice were carried out. Two hybridoma cell lines that recognized the immunogen D. bryoniae were obtained. One monoclonal antibody (MAb), Dbl, recognized the supernatant while another MAb, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid of the two MAb, Dbl and Db15, the immunotypes of which were identified as IgG1 and IgG2b, respectively. Titers of MAb Dbl and MAb Db15 were measured and the absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with D. bryoniae but none reacted with other viral isolates, Cucumber mosaic virus and Cucumber green mottle mosaic virus. Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{-3}$/well by indirect ELISA. Characterization of the MAbs Dbl, Db15 antigen by heat and protease treatments, which suggests that the epitope recognized by these two MAbs was glycoprotein.

• The Plant Pathology Journal
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• v.15 no.5
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.625-629
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• 1998

Turnip mosaic potyviruses (TuMV) were isolated from Rorippa indica and Armoracia lapathifolia showing mosaic symptoms in field. Identification of the TuMVs were carried out by host reactions of indicator plants, electron micrograph, serological properties and reverse transcription-poly-merase chain reaction (RT-PCR). Both viruses systemically infected Chenopodium quinoa, Nicotiana clevelandii, Brassica rapa, B. campestris subsp. pekinensis, B. juncea and Raphanus sativus, and developed local infection on inoculated leaves of C. quinoa, C. amaranticola, C. album, N. tabacum cv. Xanthi nc and Gomphrena grobosa. However, the viruses did not infect on N. glutinosa, Cucumis sativus and Vigna unguiculata. The filamentous particles, about 720 nm in length, and inclusion bodies were observed from the infected leaf tissues by dipping on electron microscopy. Crude sap of leaf infected with the viruses was reacted positively with an antiserum of TuMV in agar gel double diffusion. For detection of the viruses, RT-PCR was carried out with TuMV--specfic oligonucleotide primer. The RT-PCR products, a 1,092 bp DNA fragment, were obtained from naturally infected leaves of R. indica and A. lapathifolia. In inoculation test to seven cruciferous weeds with TuMV, infection occurred in Arabis glabra, Barbarea orthoceras, Capsella bursa-pastoris, Draba nomorosa var. hebecarpa, Rorippa cantoniensis and Thlaspi arvense.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.171-182
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• 2018

Papaya (Carica papaya L.) is one of the crops widely planted in tropical and subtropical areas. The papaya fruit has low calories and are plentiful in vitamins A and C and in minerals. A major problem in papaya production is a plant disease caused by the papaya ringspot virus (PRSV). The first PRSV-resistant GM papaya expressing a PRSV coat protein gene was developed by USA scientists in 1992. The first commercial GM papaya cultivars derived from the event was approved by the US government in 1997. Development of transgenic papayas has been focused on vaccine production and limited agricultural traits, including insect and pathogen resistance, long shelf life, and aluminum and herbicide tolerance. Approximately 17 countries, including the USA and China, produced transgenic papayas and/or commercialized them, which provoked studies on biosafety assessment and development of GM-detection technologies. For the biosafety assessment of potential effects on human health, effects of long-term feeding to model animals have been studied in terms of toxicity and allergenicity. Studies on environmental safety assessment include influence on soil-microbial biodiversity and transfer to soil bacteria of GM selection markers. Many countries, such as Korea, the European Union, and Japan, that have strict regulations for GM crops have serious concerns about unintended introduction of GM cultivars and food commodities using unauthorized GM crops. Transgene- and/or GM event-specific molecular markers and technologies for genomics-based detection of unauthorized GM papaya have been developed and have resulted in the robust detection of GM papayas.

• Korean Journal of Poultry Science
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• v.44 no.2
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• pp.93-102
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• 2017

A duplex RT-PCR (dRT-PCR) assay was developed for the simultaneous detection and discrimination of non-virulent and virulent Newcastle disease virus (NDV) in a single PCR tube. Primers targeting the large polymerase protein (L) gene and the fusion protein (F) gene of NDV were designed to detect all NDVs (by common type PCR primers) and virulent NDVs (by pathotype PCR primers), respectively and evaluated experimentally with reference NDV strains and other poultry viral pathogens. PCR products of the expected size of 386 bp were amplified from all NDV samples whereas PCR products of the expected size of 229 bp were amplified from virulent NDV samples alone. Cross reaction was not observed with other avian viral pathogens. The detection limit of NDV by the dRT-PCR was estimated to be $10^3$ 50% egg infectious dose/0.1 mL. In the dRT-PCR using field isolates of NDV, the pathotype PCR primers detected specifically all of virulent field isolates of NDV from Malaysia, Pakistan and China whereas common type PCR primers detected 94.4% (51/54) of field isolates of NDV from China. Three Chinese NDV isolates with false negative result were non-virulent viruses. Our results indicate that the dRT-PCR might provide a rapid and simple tool for rapid simultaneous detection and discrimination of non-virulent and virulent NDVs. Therefore the developed dRT-PCR assay provides a powerful novel means for the rapid diagnosis of Newcastle disease.

• Korean Journal of Veterinary Service
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• v.39 no.2
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

• Korean Journal of Veterinary Service
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• v.37 no.4
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• pp.263-271
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• 2014

In this study, we developed a cost and time saving one-step multiplex RT-PCR for the simultaneous detection and differentiation of swine influenza viruses (SIV) and 2009 pandemic influenza H1N1 virus (pH1N1). The one-step multiplex RT-PCR using four sets of primer was confirmed to be capable of detection of all SIV subtypes and differential diagnosis of major SIV subtype H1, H3 and pH1N1 on individual or mixed viral culture samples. The sensitivity of the multiplex RT-PCR was determined to be at least $2^{-6}$ $HA/25{\mu}L$ of the presented SIVs, providing sufficient efficacy for a routine SIV monitoring in diagnostic laboratories. In addition, compared with the conventional RT-PCR methods that cannot avoid the carryover DNA contamination, the developed RT-PCR applied with the uracil DNA glycosylase (UNG) system was proven to prevent a false positive reaction by carryover contamination of the pre-amplified DNA. In conclusion, the one-step RT-PCR with UNG system could be applicable to detect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.