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RT-PCR Detection of Citrus Tristeza Virus form Early Satsuma Nandarin and Yuzu in Cheju Island

  • Kim, Daehyun;Jaewook Hyun;Hyunsik Hwang;Lee, Sukchan
    • The Plant Pathology Journal
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    • v.16 no.1
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    • pp.48-51
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    • 2000
  • Citrus tristeza virus (CTV) was identified form CTV-infected early satsuma mandarin (Citus unshiu) and yuzu (C.junos) by RT-PCR. The total RNAs were isolated from citrus bark and seaf tissues infected with CTV and reverse transcription was followed with primers designed for amplifying CTV coat protein gene. DNA fragments 738 bp were amplified by RT-PCR and these products were colned for sequence analysis. Based on the sequence analysis, this PCR product has 97% sequence homology to CTV (T-385) CP gene isolated from USA. RT-PCR assay for CTV detection was more sensitivity than ELISA assay which was done with anti-CTV CP antibody. This is the frist report about CTV identification in Cheju island Korea.

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Comparison of CT based-CTV plan and CT based-ICRU38 plan in brachytherapy planning of uterine cervix cancer (자궁경부암 강내조사 시 CT를 이용한 CTV에 근거한 치료계획과 ICRU 38에 근거할 치료계획의 비교)

  • Shim JinSup;Jo JungKun;Si ChangKeun;Lee KiHo;Lee DuHyun;Choi KyeSuk
    • The Journal of Korean Society for Radiation Therapy
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    • v.16 no.2
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    • pp.9-17
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    • 2004
  • Purpose : Although Improve of CT, MRI Radio-diagnosis and Radiation Therapy Planing, but we still use ICRU38 Planning system(2D film-based) broadly. 3-Dimensional ICR plan(CT image based) is not only offer tumor and normal tissue dose but also support DVH information. On this study, we plan irradiation-goal dose on CTV(CTV plan) and irradiation-goal dose on ICRU 38 point(ICRU38 plan) by use CT image. And compare with tumor-dose, rectal-dose, bladder-dose on both planning, and analysis DVH Method and Material : Sample 11 patients who treated by Ir-192 HDR. After 40Gy external radiation therapy, ICR plan established. All the patients carry out CT-image scanned by CT-simulator. And we use PLATO(Nucletron) v.14.2 planing system. We draw CTV, rectum, bladder on the CT image. And establish plan irradiation-$100\%$ dose on CTV(CTV plan) and irradiation-$100\%$ dose on A-point(ICRU38 plan) Result : CTV volume($average{\pm}SD$) is $21.8{\pm}26.6cm^3$, rectum volume($average{\pm}SD$) is $60.9{\pm}25.0cm^3$, bladder volume($average{\pm}SD$) is $116.1{\pm}40.1cm^3$ sampled 11 patients. The volume including $100\%$ dose is $126.7{\pm}18.9cm^3$ on ICRU plan and $98.2{\pm}74.5cm^3$ on CTV plan. On ICRU planning, the other one's $22.0cm^3$ CTV volume who residual tumor size excess 4cm is not including $100\%$ isodose. 8 patient's $12.9{\pm}5.9cm^3$ tumor volume who residual tumor size belows 4cm irradiated $100\%$ dose. Bladder dose(recommended by ICRU 38) is $90.1{\pm}21.3\%$ on ICRU plan, $68.7{\pm}26.6\%$ on CTV plan, and rectal dose is $86.4{\pm}18.3\%,\;76.9{\pm}15.6\%$. Bladder and Rectum maximum dose is $137.2{\pm}50.1\%,\;101.1{\pm}41.8\%$ on ICRU plan, $107.6{\pm}47.9\%,\;86.9{\pm}30.8\%$ on CTV plan. Therefore CTV plan more less normal issue-irradiated dose than ICRU plan. But one patient case who residual tumor size excess 4cm, Normal tissue dose more higher than critical dose remarkably on CTV plan. $80\%$over-Irradiated rectal dose(V80rec) is $1.8{\pm}2.4cm^3$ on ICRU plan, $0.7{\pm}1.0cm^3$ on CTV plan. $80\%$over-Irradiated bladder dose(V80bla) is $12.2{\pm}8.9cm^3$ on ICRU plan, $3.5{\pm}4.1cm^3$ on CTV plan. Likewise, CTV plan more less irradiated normal tissue than ICRU38 plan. Conclusion : Although, prove effect and stability about previous ICRU plan, if we use CTV plan by CT image, we will reduce normal tissue dose and irradiated goal-dose at residual tumor on small residual tumor case. But bigger residual tumor case, we need more research about effective 3D-planning.

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Detection of Citrus Tristeza Virus by RT-PCR and Status of CTV Infection among Citrus Trees in Cheju Island

  • Oh, Hyun-Jeong;Park, Sung-Hugh;Lee, Se-Yong;Jeon, Gyeong-Lyong;Riu, Key-Zung;U, Zanh-Kual
    • The Plant Pathology Journal
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    • v.15 no.6
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    • pp.335-339
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    • 1999
  • Citrus tristeza virus(CTV), an aphid-borne closterovirus, is one of the most destructive pathogens of citrus. It has caused rapid decline in growth, stem pitting and death in citrus trees. A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for detection of CTV and investigation of the CTV infection status of citrus and its related cultivars in Cheju island. For RT-PCR based CTV detection, primers were designed to amplify 670bp of coat protein gene. A screening test for CTV in citrus cultivars was conducted from March to July in 1999. Seventy individual citrus trees representing 9 species of 3 genera were tested. The infection rates of CTV for leaves from the years or older trees of late maturing citrus varieties such as Yuzu (C. junos Sieb. ex Tanaka), Navel orange (C.sinensis Osbeck), Kiyomitanger (C. unshiu x C. sinensis), and Shiranuhi ((C. unshiu x C. sinensis) x C. reticulata) were 100%, 80%, 60%, and 60% respectively. The CTV infection rates in Early satsuma mandarins such as 'Miyagawa Early' Satsuma mandarins (C. unshiu Marc. var. Miyagawa) and 'Okitsu Early' Satsuma mandarins (C. unshiu Marc. var. Okitsu) were 100%, and 60%, respectively. CTV was not detected in Cheju native Dangyooja (C. unshiu Marc. var. Osbeck), Trifoliate orange (Poncirus trifoliata) and Kumquat (Fortunella margarita Swingle). In conclusion, RT-PCR assay can be successfully applied to the detection of CTV in citrus trees.

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Sequence Analysis of the Coat Protein Gene of Citrus Tristeza Virus Isolated form Cheju Island

  • Park, Hyoun-Hyang;Kim, Dae-Hyun;Hyun, Woo-Taek;Moon, Doo-Khil;Koh, Young-jin;Park, Tae-Jin
    • The Plant Pathology Journal
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    • v.16 no.1
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    • pp.43-47
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    • 2000
  • Citrus tristeza virus (CTV) is the causal agent of one of the most important diseases of citrus. Recently, CTV has been detected in Cheju Island by ELISA. The coat protein (CP) gene of CTV isolated form Cheju Island was cloned by RT-PCR and the nucleotide was analyzed in this study. Citrus leaves were collected from trees showing decline symptoms from various region of Cheju Island in the summer of 1998 and 1999. The CP gene open reading frame is composed of 670 nucleotides and encodes a polypeptide of 223 amono acids. Sequence analysis the CP gene revealed that two CTV strains present in Cheju Island. Viruses collected form Sogwipo area and Cheju City area in 1999 ahowed 91-93% nucleotide sequence homology with CTV T36 strain. Viruses collected form Cheju City area in 1999 and Sogwipo City in 1998 showed 94-98% nucleotide sequence homology with CTV SY568 strain. A efficient viral RNA extraction methods was developed by modifying procedure for animal virus RNA purification methods and PCR product was detected form one tenth of RNA purified from as small as 45 mg fresh or frozen tissue.

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Comparison of CT based-CTV plan and CT based-ICRU38 plan in Brachytherapy Planning of Uterine Cervix Cancer (자궁경부암 강내조사 시 CT를 이용한 CTV에 근거한 치료계획과 ICRU 38에 근거한 치료계획의 비교)

  • Cho, Jung-Ken;Han, Tae-Jong
    • Journal of Radiation Protection and Research
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    • v.32 no.3
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    • pp.105-110
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    • 2007
  • Purpose : In spite of recent remarkable improvement of diagnostic imaging modalities such as CT, MRI, and PET and radiation therapy planing systems, ICR plan of uterine cervix cancer, based on recommendation of ICRU38(2D film-based) such as Point A, is still used widely. A 3-dimensional ICR plan based on CT image provides dose-volume histogram(DVH) information of the tumor and normal tissue. In this study, we compared tumor-dose, rectal-dose and bladder-dose through an analysis of DVH between CTV plan and ICRU38 plan based on CT image. Method and Material : We analyzed 11 patients with a cervix cancer who received the ICR of Ir-192 HDR. After 40Gy of external beam radiation therapy, ICR plan was established using PLATO(Nucletron) v.14.2 planing system. CT scan was done to all the patients using CT-simulator(Ultra Z, Philips). We contoured CTV, rectum and bladder on the CT image and established CTV plan which delivers the 100% dose to CTV and ICRU plan which delivers the 100% dose to the point A. Result : The volume$(average{\pm}SD)$ of CTV, rectum and bladder in all of 11 patients is $21.8{\pm}6.6cm^3,\;60.9{\pm}25.0cm^3,\;111.6{\pm}40.1cm^3$ respectively. The volume covered by 100% isodose curve is $126.7{\pm}18.9cm^3$ in ICRU plan and $98.2{\pm}74.5cm^3$ in CTV plan(p=0.0001), respectively. In (On) ICRU planning, $22.0cm^3$ of CTV volume was not covered by 100% isodose curve in one patient whose residual tumor size is greater than 4cm, while more than 100% dose was irradiated unnecessarily to the normal organ of $62.2{\pm}4.8cm^3$ other than the tumor in the remaining 10 patients with a residual tumor less than 4cm in size. Bladder dose recommended by ICRU 38 was $90.1{\pm}21.3%$ and $68.7{\pm}26.6%$ in ICRU plan and in CTV plan respectively(p=0.001) while rectal dose recommended by ICRU 38 was $86.4{\pm}18.3%$ and $76.9{\pm}15.6%$ in ICRU plan and in CTV plan, respectively(p=0.08). Bladder and rectum maximum dose was $137.2{\pm}50.1%,\;101.1{\pm}41.8%$ in ICRU plan and $107.6{\pm}47.9%,\;86.9{\pm}30.8%$ in CTV plan, respectively. Therefore, the radiation dose to normal organ was lower in CTV plan than in ICRU plan. But the normal tissue dose was remarkably higher than a recommended dose in CTV plan in one patient whose residual tumor size was greater than 4cm. The volume of rectum receiving more than 80% isodose (V80rec) was $1.8{\pm}2.4cm^3$ in ICRU plan and $0.7{\pm}1.0cm^3$ in CTV plan(p=0.02). The volume of bladder receiving more than 80% isodose(V80bla) was $12.2{\pm}8.9cm^3$ in ICRU plan and $3.5{\pm}4.1cm^3$ in CTV plan(p=0.005). According to these parameters, CTV plan could also save more normal tissue compared to ICRU38 plan. Conclusion : An unnecessary excessive radiation dose is irradiated to normal tissues within 100% isodose area in the traditional ICRU plan in case of a small size of cervix cancer, but if we use CTV plan based on CT image, the normal tissue dose could be reduced remarkably without a compromise of tumor dose. However, in a large tumor case, we need more research on an effective 3D-planing to reduce the normal tissue dose.

Comparison of viral population of pathologically and geographically different areas of Southern provinces and Jeju, Korea

  • Kim, Daehyun;Hyekyung Shim;Jaewook Hyeon;Kim, Kwangsik;Lee, Sukchan
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.123.1-123
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    • 2003
  • The objective of this work was to analyze the population of sequence variants of citrus tristeza virus (CTV) isolates in Korea and to make the phylogeny trees of CTV in Korea. We also tried to analyze and find the mild strain of CTV to apply for the cross protection. The CTV isolates from yuzu (C. Junos) collected from different geographic areas of Southern provinces such as Namhae-Do, Kerche-Do, Bosung, Wan-Do and Koheung and Jeju-Bo, Korea were used for SSCP analysis. The SSCP profiles of the cDNAS obtained by RT-PCR with primers specifically designed for the p20 of the CTV population. The SSCP profiles obtained from 150 PCR products in yuzu contained two or three DNA bands, whereas, in some case, others contained four or more bands of similar intensity. The pathologically mild isolates of CTV usually yielded two DNA bands by SSCP profiles, whereas the SSCP profiles of the most virulent isolates contained more than two DNA bands. Plants shown severe stem pitting were corresponded to those plants with typical SSCP profiles of severe strains, and vice versa. This results indicate that the primers designed for SSCP analysis can be used for distinguishing the mild strains from severe strains of CTV.

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Dosimetric Evaluation of Static and Dynamic Intensity Modulated Radiation Treatment Planning and Delivery (세기조절방사선치료에서 조사방법이 빔 파라미터 및 선량에 미치는 영향에 대한 연구)

  • Kim Sung-Kyu;Kim Myung-Se;Yun Sang-Mo
    • Progress in Medical Physics
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    • v.17 no.2
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    • pp.114-122
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    • 2006
  • The two commonly used methods in delivering intensity modulated radiation therapy (IMRT) plan are the dynamic (sliding window) and static (stop and shoot) mode. In this study, the two IMRI delivery techniques are compared by measuring point dose and dose distributions. Using treatment planning system, clinical target volume (CTV) was created as a sphere with various diameter (3 cm, 7 cm, 12 cm). Two IMRT plans were peformed to deliver 200 cGy to the CTV in dynamic and static mode. The two plans were delivered on a phantom and central point dose and dose distributions were measured. The central point dose differences between static and dynamic IMRT delivery were 0.2%, 0.2% and 0.4% when the diameter of CTV was 3 cm, 7 cm, and 12 cm, respectively. The differences In volume receiving 90% of the proscribed dose were 2.7%, 2.2%, and 2.9% for the diameter of CTV was 3 cm, 7 cm, and 12 cm, respectively. For lung cancer patients, the differences in central point dose were 0.2%, 0.2%, and 0.4% when the volume of CTV was 35.5 cc, 296.8 cc, and 903.5 cc, respectively. The differences in volume receiving 90% of the prescribed dose were 2.7%, 4.8%, and 9.1% when the volume of CTV was 35.5 cc, 296.8 cc, and 903.5 cc, respectively. In conclusion, it was possible to deliver IMRT plans using dynamic mode of MLC operation although the loaves are In motion during radiation delivery.

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Identification of Endogenous Genes for Normalizing Titer Variation of Citrus Tristeza Virus in Aphids at Different Post-acquisition Feeding Times

  • Wang, Hongsu;Chen, Qi;Liu, Luqin;Zhou, Yan;Wang, Huanhuan;Li, Zhongan;Liu, Jinxiang
    • The Plant Pathology Journal
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    • v.38 no.4
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    • pp.287-295
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    • 2022
  • Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.

Micrografting and Heat Treatment Combination for Eliminating Virus of CTV-infected Citrus (CTV 바이러스 보균 감귤나무로부터 열처리와 경정접목을 통한 바이러스 제거)

  • Chae, Chi Won;Yun, Su Hyun;Park, Jae Ho;Hyun, Jae Wook;Koh, Sang Wook;Lee, Dong Hoon
    • Journal of Life Science
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    • v.23 no.2
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    • pp.267-272
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    • 2013
  • This study was conducted to eliminate viruses from citrus-infected plants using micrografting and thermotherapy. Six citrus cultivars including a 'Setoka' hybrid were used as plant sources. The TAS-ELISA technique demonstrated that several plants were CTV positive. However, no CTV symptoms were detected in plants obtained from shoots and treated at a high temperature of $40^{\circ}C$ during the day and night and micrografted for two weeks with old trifoliate orange rootstock in vitro. Indexing of CTV, SDV, and CTLV for RT-PCR analysis of the eleven citrus seedlings, including 'Setoka', 'Samdajosang', 'Pungkwang', 'Shiranuhi', and 'Ehimekashi dai28go' was virus free following the micrografting and thermal therapy.

Evaluation of Setup Uncertainty on the CTV Dose and Setup Margin Using Monte Carlo Simulation (몬테칼로 전산모사를 이용한 셋업오차가 임상표적체적에 전달되는 선량과 셋업마진에 대하여 미치는 영향 평가)

  • Cho, Il-Sung;Kwark, Jung-Won;Cho, Byung-Chul;Kim, Jong-Hoon;Ahn, Seung-Do;Park, Sung-Ho
    • Progress in Medical Physics
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    • v.23 no.2
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    • pp.81-90
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    • 2012
  • The effect of setup uncertainties on CTV dose and the correlation between setup uncertainties and setup margin were evaluated by Monte Carlo based numerical simulation. Patient specific information of IMRT treatment plan for rectal cancer designed on the VARIAN Eclipse planning system was utilized for the Monte Carlo simulation program including the planned dose distribution and tumor volume information of a rectal cancer patient. The simulation program was developed for the purpose of the study on Linux environment using open source packages, GNU C++ and ROOT data analysis framework. All misalignments of patient setup were assumed to follow the central limit theorem. Thus systematic and random errors were generated according to the gaussian statistics with a given standard deviation as simulation input parameter. After the setup error simulations, the change of dose in CTV volume was analyzed with the simulation result. In order to verify the conventional margin recipe, the correlation between setup error and setup margin was compared with the margin formula developed on three dimensional conformal radiation therapy. The simulation was performed total 2,000 times for each simulation input of systematic and random errors independently. The size of standard deviation for generating patient setup errors was changed from 1 mm to 10 mm with 1 mm step. In case for the systematic error the minimum dose on CTV $D_{min}^{stat{\cdot}}$ was decreased from 100.4 to 72.50% and the mean dose $\bar{D}_{syst{\cdot}}$ was decreased from 100.45% to 97.88%. However the standard deviation of dose distribution in CTV volume was increased from 0.02% to 3.33%. The effect of random error gave the same result of a reduction of mean and minimum dose to CTV volume. It was found that the minimum dose on CTV volume $D_{min}^{rand{\cdot}}$ was reduced from 100.45% to 94.80% and the mean dose to CTV $\bar{D}_{rand{\cdot}}$ was decreased from 100.46% to 97.87%. Like systematic error, the standard deviation of CTV dose ${\Delta}D_{rand}$ was increased from 0.01% to 0.63%. After calculating a size of margin for each systematic and random error the "population ratio" was introduced and applied to verify margin recipe. It was found that the conventional margin formula satisfy margin object on IMRT treatment for rectal cancer. It is considered that the developed Monte-carlo based simulation program might be useful to study for patient setup error and dose coverage in CTV volume due to variations of margin size and setup error.