• 대한수의학회지
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• 제57권1호
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• pp.31-36
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• 2017

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT ($PRNT_{90}$). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and $PRNT_{90}$ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and $PRNT_{90}$ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

• 한국식물병리학회지
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• 제10권2호
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• pp.136-139
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• 1994

Turnip mosaic virus (TuMV) was isolated from Rorippa islandica showing mild mosaic symptom in growing field of Chinese cabbage and radish. Identification of the virus was based on host range, transmission by aphids, electron micrograph, serological reaction and hybridization detection. The virus systemically infected on Chenopodium quinoa, Nicotiana clevelandii, N. glutinosa, Brassica rapa, B. campestris subsp. pekinensis and Raphanus sativus, whereas showed local infection on C. amaranticolor, Gomphrena globosa and Tetragonia tetragonoides. The virus was transmitted by aphid (Myzus persicae). The virus particle was filamentous with 720$\times$12 nm in length, and reacted positively with an antiserum of TuMV in agar gel double duffusion test. In slot-blot hybridization using the digoxigenin(DIG)-labeled RNA probe, TuMV-RNA could be detected in sap of R. islandica infected with the virus. This is the first report of a natural infection of that virus on R. islandica.

• The Plant Pathology Journal
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• 제27권1호
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• pp.44-52
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• 2011

Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.

• 식물병연구
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• 제19권1호
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• pp.36-44
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• 2013

카네이션은 세계 3대 절화용 화훼작물의 하나로, 농가 생산액 210억 원에 이르는 주요작물이다. 이들은 절화, 종자, 묘 및 삽수의 4 품목으로 수출입 되고 있다. 카네이션과 같은 영양번식성 작물의 경우, 증식하는 과정 중에 바이러스의 확산과 전파가 용이한데, 우리나라에서는 카네이션괴저바이러스(CNFV)와 카네이션둥근반점바이러스(CRSV)를 식물검역 바이러스로 지정하여 수입검사를 수행하고 있다. 본 연구에서는 CNFV와 CRSV를 신속, 정밀하고 쉽게 진단할 수 있는 특이적인 프라이머를 고안하였으며, 높은 검출 감도를 가지는 nested 프라이머 조합을 개발하였다. CNFV를 검사하기 위해 최종 선발된 특이적인 프라이머는 2 세트로 288과 447 bp를, CRSV를 검사하기 위해 최종 선발된 특이적인 프라이머는 2 세트로 503과 549 bp를 증폭하였다. CNFV의 nested는 2 세트 모두 147 bp로 동일하며, CRSV는 각각 395와 347 bp의 밴드를 증폭하였다. 또한, 실험의 신뢰도를 높이기 위하여, 증폭산물에 염기서열 6개를 삽입한 플라스미드를 제작하여 양성대조구로 활용하였다. 본 연구에서 개발한 방법은, 향후 CNFV와 CRSV에 대한 신속, 정밀한 국경검역을 지원할 수 있을 것이라고 기대된다.

• The Plant Pathology Journal
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• 제30권4호
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• pp.416-424
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• 2014

Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

• The Plant Pathology Journal
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• 제36권2호
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• pp.170-178
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• 2020

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65℃ and 60℃ for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

• 식물병연구
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• 제23권2호
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• pp.193-201
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• 2017

국내의 토마토에서 발생하는 주요 바이러스는 Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), Tomato mosaic virus (ToMV)이다. 이들 바이러스를 진단하기 위해 프라이머 세트와 반응액을 포함하는 역전사중합반응(RT-PCR)법의 조건을 조사하였다. 공시한 바이러스에 특이적인 염기서열로부터 모두 46개 프라이머 세트를 설계하고, 이를 이용해 주형을 넣지 않은 RT-PCR에서 비특이 반응을 조사하였다. 이들 가운데 16개 조합을 건전한 토마토 RNA에 적용한 결과 프라이머 세트와 RT-PCR 반응액 간의 친화성이 비특이 반응 감소에 영향을 주었다. cDNA 합성과 관련된 인자와 RT-PCR 반응액 사이의 조합을 근거로 ToCV 진단을 위한 두 종류의 반응액을 선발하였다. ToCV 진단 시 수립된 조건을 나머지 바이러스 진단에 적용했을 때, 특이성이 높은 프라이머 세트 C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), C065 (ToMV)를 선발할 수 있었다. 이들 프라이머 세트는 공시한 바이러스를 특이적으로 진단하는 데 유용할 것으로 판단된다.

• 식물병연구
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• 제27권2호
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• pp.79-83
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• 2021

The aim of the present study was to develop a sensitive and specific detection method for the rapid detection of apple scar skin viroid (ASSVd) in apple leaves. The resulting reverse transcription recombinase polymerase amplification (RT-RPA) assay can be completed in 10 min at 42℃, is 10 times more sensitive than conventional reverse transcription polymerase chain reaction, and can specifically amplify ASSVd without any cross-reactivity with other common apple viruses, including apple stem grooving virus, apple stem pitting virus, and apple chlorotic leaf spot virus. The reliability of the RT-RPA assay was assessed, and the findings suggested that it can be successfully utilized to detect ASSVd in field-collected samples. The RT-RPA assay developed in the present study provides a potentially valuable means for improving the detection of ASSVd in viroid-free certification programs, especially in resource-limited conditions.

• 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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• 제19권3호
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• pp.220-225
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• 2013

본 연구에서는 식물검역바이러스 4종(TBRV, ArMV, CLRV 및 GFLV)을 RT-PCR과 nested PCR 방법으로 진단 할 수 있는 방법을 개발하였다. 본 연구에서 개발한 방법은 모두 같은 PCR 조건으로 검사자에게 편리성과 신속성을 높여줄 뿐 아니라, 돌연변이-양성대조구의 사용으로 실험 오염여부를 확인할 수 있어 더욱 정확하다. 개발한 방법으로 최근 3년 Nepovirus속 4종의 바이러스를 검사한 결과, 27건을 검출하여 검역처분 하였다. 본 연구 결과들은 앞으로도 수출입 식물에서 해당 바이러스들을 신속, 정밀하게 진단할 수 있는 방법으로 활용할 수 있을 것으로 기대된다.