• 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.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.821-831
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• 2022

American plum line pattern virus (APLPV), a member of the genus Ilarvirus in the family Bromoviridae, is one of the plant quarantine pathogens in Korea. In this study, 15 candidate primer sets were designed and examined to develop a reverse transcription polymerase chain reaction (RT-PCR) assay for plant quarantine inspection of APLPV. Using APLPV-infected and healthy samples, the primer sets were assessed for APLPV detection. To confirm the occurrence of nonspecific reactions, six ilarviruses (Apple mosaic virus, Asparagus virus 2, Blueberry shock virus, Prune dwarf virus, Prunus necrotic ringspot virus, and Tobacco streak virus) and 10 target plants (Prunus mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera) were examined. Finally, two primer sets were selected. These primer sets could generate the expected amplicons even with at least 1 ng of the total RNA template in concentration-dependent amplifications. In addition, a positive clone was developed for use as a positive control in the abovementioned RT-PCR assay.

• Parasites, Hosts and Diseases
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• v.35 no.3
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• pp.203-210
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• 1997

The difrrrenlial display reverse transcription polymerase chain reaction (DDRT-PCR) aniilysis roils performed to identify the pathogellir strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenif strain YS-27 and the non-pathogenic strain S 16. respectively. Three kinds of rirsl stranded rDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT 11M (M: A. C, or G) primers. Each cDNA lemplatr was used for DDRT-PCK analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oli해-dT11M primers. Of these 31 amplit'tons were verified as the amplirons amplified only from the mRNAs of the pathogenic strain by DNA slots biol llybridizatioil. Furthel cklaracleization of the 31 pathogenic strain sprcifil amplicons by DNA slot blot hybridlnation analysis using biotin labeled Probes or the PCR amplified DNA of rysteine proteinase genes revealed that 21 of them were amplliried from the maNAs of the cysteine proteinase genes. Four randomly selected amplirons out of the rest 10 amplirons were used fur screening of cDNA library followed by immunoscreening and all of them were turned outs to be amplified from the mRNA.

• Journal of Plant Biology
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• v.38 no.3
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• pp.267-274
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• 1995

Based upon the nucleotide sequence of As strain of cucumber mosaic virus (CMV-As0 RNA4, coat protein (CP) gene was selected for the design of oligonucleotide primers of polymerase chain reaction (PCR) for detection and identification of the virus. Reverse transcription and polymerase chain reaction (RT-PCR) was performed with a set of 18-mer CMV CP-specific primers to amplify a 671 bp fragment from crude nucleic acid extracts of virus-infected leaf tissues as well as purified viral RNAs. The minimum concentrations of template viral RNA and crude nucleic acids from infected tobacco tissue required to detect the virus were 1.0 fg and 1:65,536 (w/v), respectively. No PCR product was obtained when potato virus Y-VN RNA or extracts of healthy plants were used as templates in RT-PCR using the same primers. The RT-PCR detected CMV-Y strain as well as CMV-As strain. Restriction analysis of the two individual PCR amplified DNA fragments from CMV-As and CMV-Y strains showed distinct polymorphic patterns. PCR product from CMV-As has a single recognition site for EcoRI and EcoRV, respectively, and the product from CMV-Y has no site for EcoRI or EcoRV but only one site for HindIII. The RT-PCR was able to detect the virus in the tissues of infected pepper, tomato and Chinese cabbage plants.

• Korean Journal of Veterinary Research
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• v.55 no.3
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• pp.185-189
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• 2015

Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012, and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conducted serosurveillance using sera obtained from seven Korean farms and optimized a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect bPIV5. The overall seropositive rate for Korean cattle was 21.4% (163/760). A farm located near the city of Milyang in Gyeoungnam province had a markedly elevated seropositive rate for bPIV5 compared to that of the other six farms. The regional seropositive rates were 4.2% (8/192) for Haman, 19.5% (18/55) for Hwasung, 73.9% (65/88) for Milyang, 26.0% (50/192) for Namwon, 1.0% (1/96) for Uljin, 13.5% (13/96) for Yeongju, and 32.7% (8/41) for Yongin. The sensitivity and specificity of three RT-PCR primer sets used to amplify the conserved fusion gene of bPIV5 were also evaluated. An RT-PCR assay using the bPIVFR3 primer set was 10-fold more sensitive than the assays using the two other primer sets and did not result in non-specific amplification. These results demonstrated that the bPIFR3 primer set can be used to detect bPIV5.

• Genomics & Informatics
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• v.18 no.4
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• pp.40.1-40.8
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• 2020

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse-transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.

• 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.

• Korean Journal of Environmental Biology
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• v.22 no.1
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• pp.206-212
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• 2004

In an effort to develop the tools for monitoring the contamination of xenoestrogen in the aquatic environment of Korea, reverse transcription-polymerase chain reaction (RT-PCR) analysis of vitellogenin (VTG) mRNA expression were optimized in Synechogobius hastus. Based on the partial VTG cDNA sequence VTG mRNA level in livers from male fishes was analyzed by RT-PCR. As an internal control beta actin mRNA was amplified. 3 ${\mu}g$ of total RNA was reverse transcribed in 20 $\mu$l reaction using murine leukemia virus 〔MuLV〕 reverse transcriptase. Subsequent PCR using the 1 ${\mu}g$ of cDNA resulted in linear increase in PCR product of VTG in female liver cDNA from 10 to 30 cycles of amplification. On the contrary, in male, PCR product first detected at 28 cycles of amplification and linearly increased during 38 cycles of amplification, suggesting that male S. hastus expresses minute amount of VTG mRNA which is $2^{-18}$ equivalent of female. In conclusion, the optimized protocol of VTG mRNA expression in the liver of male S. hastus will be promising the environmental monitoring the xenoestrogen contamination in the western coast and estuaries in Korea.

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• Journal of Apiculture
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• v.32 no.3
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• pp.171-180
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• 2017

Slow Bee Paralysis Virus (SBPV) is a pathogenic virus against honeybee and bumblebee, causes the death of adult bee by paralyzing the fore-leg of bee. In this study, for rapid detection of SBPV from bumblebee, SBPV-specific Ultra-rapid Reverse transcription PCR was developed. After optimizing of SBPV-specific Ultra-rapid PCR, the existence of $1.0{\times}10^8$ SBPV-specific DNA molecules could be recognized in 3 minute and 35 seconds. Even $1.0{\times}10^1$ molecules of SBPV-specific DNA could be measured with quantitative manner. Meanwhile, from both imported bumblebee and bumblebee produced in Korea, SBPV were detected using proposed method. In the laboratory as well as in the field, SBPV-specific Ultra-rapid Reverse transcription PCR would be applied and might be expected as useful tools at production of bumblebee or inspection for the import and export system of bumblebee.