[논문]Development of Single-tube Multiplex Immunocapture RT-PCR Assay for Simultaneous Detection of Two Pepper Tobamoviruses

Kim, Jae-Hyun; Choi, Gug-Seoun; Kim, Jeong-Soo; Lee, Sin-Ho; Choi, Jang-Kyung; Ryu, Ki-Ryun

doi:10.5423/ppj.2006.22.2.164

Development of Single-tube Multiplex Immunocapture RT-PCR Assay for Simultaneous Detection of Two Pepper Tobamoviruses 원문보기

The plant pathology journal, v.22 no.2, 2006년, pp.164 - 167

Kim, Jae-Hyun (Bio-resources, Research Group, KT&G Central Research Institute) , Choi, Gug-Seoun (National Horticultural Research Institute, Rural Development Administration) , Kim, Jeong-Soo (National Horticultural Research Institute, Rural Development Administration) , Lee, Sin-Ho (National Horticultural Research Institute, Rural Development Administration) , Choi, Jang-Kyung (Division of Biological Environment, Kanwon National University) , Ryu, Ki-Ryun (Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women's University)

Abstract ▼ AI-Helper

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

주제어

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

문제 정의

This study was conducted .to develop an IC/RT-PCR assay for simultaneous detection of PMMoV and TMGMV in pepper that would improve both detection sensitivity and specificity compare to the ELISA systems currently in use.

제안 방법

occidentalis. For this study, single and mixed infection with PMMoV and TMGMV were conducted with carborundum-dusted Capsicum annuum Tary Calwonder5 in a green house at 25±2℃. Test samples consisted of two types: (i) crude extracts prepared from fresh tissue, and (ii) dilutions of purfied virus.
In this study, we have developed a multiplex IC/RT-PCR for the simultaneous detection of both PMMoV and TMGMV and compared the sensitivity and specificity of the IC/RT-PCR approach with that of ELISA systems currently in use.

대상 데이터

The primer sequences were based on the published sequence of genomes of PMMoV-Kr (Yoon, 2003) and TMGMVKP (Choi et al, 2002). The primers used for the specific detection of PMMoV and TMGMV were PM317-F 5f-ACA GTT TCC AGT GCC AAT CA-3; PM317-R 5'-AAG CGT CTC GGC AGT TG-3' (flanking a 317 base position of the coat protein of PMMoV), and CPTMG-F 5'-TCG AGT ACG TTT TAA TCA AT-3' and CPTMG-R 5'-ATT [TA GGA AAT CTC ACA AC-3' (flanking a 510 base position of the coat protein of TMGMV). Tubes were spun briefly, reverse transcriptase (RT) reaction was carried out with on cycle at 42℃ for 45 mins and 35 cycles of PCR amplication using the step program (95℃, 45 sec.

이론/모형

Immunocapture (IC) was carried out using a coating and trapping method adapted from Clack and Adams (1977). Micro tubes (200 μ1) were coated with antibodies (50 μ1) to both PMMoV and TMGMV at concentrations of 1 mg/ml each.

참고문헌 (23)

Candresse, T., Lanneau, M., Revers, F., Macquaire, G., German, S., Dunez, J., Grasseau, N. and Malinovsky, T. 1995a. An immunocapture PCR assay adapted to the detection and the analysis of the morecular variability of Apple chlorotic leaf spot virus. Acta Hort. 386:136-147
Choi, G. S., Kim, J. H., Lee, D. H., Kim, J. S. and Ryu, K. H. 2005. Occurrence and distribution of viruses infecting pepper in Korea. Plant Pathol. J. 21 :258-261

원문보기 상세보기
Choi, G. S., Kim, J. H., Ryu, K. H., Choi, J. K., Chae, S. Y., Kim, J. S., Chung, B. N., Kim, H. R. and Choi, Y. M. 2002. Frist report of Tobacco mild green mosaic virus infecting pepper in Korea. Plant Pathol. J. 18: 323-327

원문보기 상세보기
Clark, M. F. and Adams, N. 1977. Characteristics of the microplate method of enzyme-linked immunoassay (ELISA) for the detection of plant viruses. J. Gen. Virol.34:475-483

상세보기
Cohen, J., Rosner, A. Kagan, S. Lampel, S., Beckelman, H., Maslenin, L., Zeidan, M. and Gera, A. 2002. A new disease in Tabemamontana caused by Tobacco mild green mosaic virus. Acta Hort. 569: 103-109
Gibb, A. J. 1988. Tobacco mild green mosaic virus. Plant Viruses Online: Description and Lists from the ηDE Database. Green, S. K. and Kim, J. S. 1991. Characteristics and control of viruses infecting peppers: a literature review. AVRDC Technical Bull. No. 18:60
Green, S. K. and Kim, J. S. 1991. Characteristics and control of viruses infecting peppers: a literature review. AVRDC Thchnical Bull. No. 18:60
Grotzinger, T. and Will, H. 1992. Sensitive methods for identification of minor hepatitis B mutant viruses. Virology 187:383-387

상세보기
Jacobi, V. and Castello, J. D. 1991. Isolation of tomato mosaic virus from water draning forest stands in New York State. Phytopathology 81: 1112-1117

상세보기
Jacobi, V., Bachand, G D., Hamelin, R. C. and Castello, J. D. 1997. Development of immunocapture PCR assay for tobacco and Tomato mosaic tobamoviruses. Phytopathology, 87:S46 (Abstract)
Jacobi, V., Bachand, G. D., Hamelin, R. C. and Castello, J. D. 1998. Development of multiplex immunocapture RT-PCR assay for detection and differentiation of Tobacco mosaic tobamoviruses. J. Virol. Methods 74:167-178

상세보기
Kang, K. Y., Choi, J. I. and La, Y. J. 1973. Isolation and identification of virus affecting pepper in Korea. J. Kor. Soc. Horti. Sci. 13:35-44
Komuro, Y. and Iwaki, M. 1969. Presence of Tobacco mosaic virus in roots of field-grown tomato plants healthy in appearance. Ann. Phytopathol. Soc. Jpn. 35:294-298

상세보기
Lanter, J. M., McGuire, J. M. and Goode, M. J. 1982. Presistence of Tomato mosaic virus in tomato debris and soil under field conditions. Plant Dis. 66:552-555

상세보기
Nassuth, A., Pollari, E., Helmeczy, K., Stewart, S. and Kofalvi, S. 2000. Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extract. J. Virol. Methods 90:37-49

상세보기
Nolasco, G., de BIas, C., torres, V. and Ponz, F.1993. A methods combining immunocapture and PCR amplification in a microtiter plat for the detection of plant viruses and subviral pathogens. J. Viral. Methods 45:201-218

상세보기
Osiowy, C. 1998. Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimes by a multiplex reverse transcription-PCR assay. J. Clin. Microbiol. 36:3149-3154

상세보기
Pappu, S. S., Brand, R., Papuu, H. R., Rybicki, E. P., Gough, K. H., Frenkel, M. J. and Niblett, C. L. 1993. A polymerase chain reaction methos adapted for selective amplification and cloning of 3'sequences of potyviral genomes: application to Dasheen mosaic virus. J. Viral. Methods 41:9-20

상세보기
Rosenfield, S. I. and Jaykus, L. A. 1999. A multiplex reverse transcription polymerase chain reaction method for the detection of foodbome viruses. J. Food Prot. 62:1210-1214

상세보기
Rowhani, A., Maningas, M. A., Lile, L. S., Daubert, S. D. and Golino, D. A.1995. Development of a detection system for viruses of woody plants based on PCR analysis of irnmobilized virions. Phytopathology 85:347-352

상세보기
Sharman, M., Thomas, J. E. and Dietzgen, R. G 2000. Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J. Viral. Methods 89:75-88

상세보기
Wetzel, T., Candress, T., Macquaire, G, Ravelonandro, M. and Dunez, J. 1992. A highly sensitive immunocapture polymerase chain reaction method for Plum pox potyvirus detection. J. Viral. Methods 39:27-37

상세보기
Yoon, J. Y. 2003. Molecular characterization of symptom attenuation of Pepper mild mottle virus. Ph.D. Thesis of Seoul Women's University, Korea

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증