[논문]Development of a Multiplex PCR for Simultaneous Detection of Blueberry Red Ringspot Virus and Blueberry Scorch Virus Including an Internal Control

Hae Min Lee; Eun Gyeong Song; Ki Hyun Ryu

doi:10.5423/rpd.2023.29.1.94

Development of a Multiplex PCR for Simultaneous Detection of Blueberry Red Ringspot Virus and Blueberry Scorch Virus Including an Internal Control 원문보기

Research in plant disease = 식물병연구, v.29 no.1, 2023년, pp.94 - 99

Hae Min Lee (Plant Virus GenBank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University) , Eun Gyeong Song (Plant Virus GenBank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University) , Ki Hyun Ryu (Plant Virus GenBank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University)

Abstract ▼ AI-Helper

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

주제어

표/그림 (5)

표 Table 1. Primers used in this study
그림 Fig. 1. Agarose gel analysis of products obtained using the monoplex polymerase chain reaction (PCR) assays of 10-fold serial dilutions of the corresponding recombinant plasmids carrying the indicated amplicons. M, 1 kb plus DNA ladder; lanes 1 to 8, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of plasmid, respectively; lane 9, negative control (empty vector); BRRSV, blueberry red ringspot virus; BlScV, blueberry scorch virus. Red arrows indicate the location of PCR amplicons of the expected sizes.
그림 Fig. 2. Agarose gel analysis of products obtained using the multiplex polymerase chain reaction (PCR) assays of the two viruses using 10-fold serial dilutions of an equal mixture of the three different recombinant plasmids, including the 18S rRNA plasmid (18S). M, 1 kb plus DNA ladder; lanes 1 to 8, products from template mixtures containing 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of each plasmid, respectively; lane 9, negative control (empty vector); BRRSV, blueberry red ringspot virus; BlScV, blueberry scorch virus. Red arrows indicate the location of PCR amplicons of the expected two viruses and 18S rRNA.
그림 Fig. 3. Monoplex polymerase chain reaction (PCR) assays for the two viruses in 14 blueberry (Vaccinium corymbosum) cultivars. The PCR products to detect blueberry red ringspot virus (BRRSV) (A) and blueberry scorch virus (BlScV) (B), including 18S used as internal control, are displayed. M, 1 kb plus DNA ladder; positive control (each target plasmid); negative control (empty vector).
그림 Fig. 4. Multipelx polymerase chain reaction assay for the simultaneous detection of the two viruses in 14 blueberry (Vaccinium corymbosum) cultivars. M, 1 kb plus DNA ladder; positive control (a mix of three plasmid templates); negative control (empty vectors); BRRSV, blueberry red ringspot virus; BlScV, blueberry scorch virus.

참고문헌 (25)

Abade Dos Santos, F. A., Carvalho, C. L., Peleteiro, M. C., Parra, F. and？Duarte, M. D. 2021. A versatile qPCR for diagnosis of leporid？gammaherpesvirus 5 using Evagreen or Taqman technologies.？Viruses 13: 715.
Abarshi, M. M., Mohammed, I. U., Jeremiah, S. C., Legg, J. P., Kumar,？P. L., Hillocks, R. J. et al. 2012. Multiplex RT-PCR assays for the？simultaneous detection of both RNA and DNA viruses infecting？cassava and the common occurrence of mixed infections by？two cassava brown streak viruses in East Africa. J. Virol. Methods？179: 176-184.

상세보기
Banerjee, S., Nayik, G.A., Kour, J. and Nazir, N. 2020. Blueberries. In:？Antioxidants in Fruits: Properties and Health Benefits, eds. by G.？A. Nayik and A. Gull, pp. 593-614. Springer, Singapore.
Bristow, P. R., Martin, R. R. and Windom, G. E. 2000. Transmission,？field spread, cultivar response, and impact on yield in highbush？blueberry infected with blueberry scorch virus. Phytopathology？90: 474-479.

상세보기
Chang, B. S. and Mahoney, R. R. 1995. Enzyme thermostabilization？by bovine serum albumin and other proteins: evidence for hydrophobic interactions. Biotechnol. Appl. Biochem. 22: 203-214.

상세보기
Chang, L., Zhang, Z., Yang, H., Li, H. and Dai, H. 2007. Detection of？strawberry RNA and DNA viruses by RT-PCR using total nucleic？acid as a template. J. Phytopathol. 155: 431-436.
Cho, I. S., Chung, B. N., Cho, J. D., Choi, G. S. and Lim, H. S. 2012. First？report of blueberry red ringspot virus infecting highbush blueberry in Korea. Plant Dis. 96: 1074.

상세보기
Ciuffo, M., Pettiti, D., Gallo, S., Masenga, V. and Turina, M. 2005. First？report of blueberry scorch virus in Europe. Plant Pathol. 54: 565.

상세보기
Dayaram, A., Opong, A., Jaschke, A., Hadfield, J., Baschiera, M., Dobson, R. C. et al. 2012. Molecular characterisation of a novel cassava associated circular ssDNA virus. Virus Res. 166: 130-135.

상세보기
Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. and Ball, L.？A. 2005. Virus Taxonomy: 8th Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San？Diego, CA, USA. 1162 pp.
Fuchs, M., Abawi, G. S., Marsella-Herrick, P., Cox, R., Cox, K.D., Carroll, J.？E. et al. 2010. Occurrence of tomato ringspot virus and tobacco？ringspot virus in highbush blueberry in New York state. J. Plant？Pathol. 92: 451-459.
Henderson, D. C. and Hammond, J. 2013. CKC: isolation of nucleic？acids from a diversity of plants using CTAB and silica columns.？Mol. Biotechnol. 53: 109-117.

상세보기
Isogai, M., Ishii, K., Umemoto, S., Watanabe, M. and Yoshikawa, N.？2009. First report of blueberry red ringspot disease caused by？blueberry red ringspot virus in Japan. J. Gen. Plant Pathol. 75:？140-143.

상세보기
Jevremovic, D., Leposavic, A. and Paunovic, S. 2015. First report of？blueberry mosaic-associated virus in highbush blueberry in？Serbia. J. Plant Pathol. 97: 541.
Kalinowska, E., Paduch-Cichal, E. and Chodorska, M. 2013. First report of Blueberry scorch virus in elderberry in Poland. Plant Dis.？97: 1515.

상세보기
Martin, R. R., Polashock, J. J. and Tzanetakis, I. E. 2012. New and？emerging viruses of blueberry and cranberry. Viruses 4: 2831-2852.

상세보기
Ozay, B. and McCalla, S. E. 2021. A review of reaction enhancement？strategies for isothermal nucleic acid amplification reactions.？Sens. Actuator Rep. 3: 100033.
Park, C. H., Song, E. G. and Ryu, K. H. 2021. A multiplex PCR assay for？the simultaneous detection of five potexviruses infecting cactus plants using dual-priming oligonucleotides (DPOs) primers.？J. Virol. Methods 298: 114280.

상세보기
Petrzik, K., Pribylova, J., Plesko, I. M. and Spak, J. 2011. Complete genome sequences of blueberry red ringspot virus (Caulimoviridae) isolates from the Czech Republic and Slovenia. Arch. Virol.？156: 1901-1903.

상세보기
Plant Quarantine Technology Center in Animal, Plant and Fisheries？Quarantine and Inspection Agency. 2013. List of plant quarantine viruses in Korea in newly revised in 2013. Res. Plant Dis. 19:？67-75. (In Korean)
Saad, N., Olmstead, J. W., Jones, J. B., Varsani, A. and Harmon, P. F.？2021. Known and new emerging viruses infecting blueberry.？Plants (Basel) 10: 2172.

상세보기
Song, H.-N., Park, M.-S., Youn, H.-S., Park, S.-J. and Hogstrand, C.？2014. Nutritional compositions and antioxidative activities of？two blueberry varieties cultivated in South Korea. Korean J.？Food Preserv. 21: 790-798. (In Korean)
Tang, K. F. J. and Lightner, D. V. 2000. Quantification of white spot？syndrome virus DNA through a competitive polymerase chain？reaction. Aquaculture 189: 11-21.

상세보기
Wegener, L. A., Punja, Z. K. and Martin, R. R. 2004. First report of？blueberry scorch virus in cranberry in Canada and the United？States. Plant Dis. 88: 427.

상세보기
Xie, L. X., Zheng, S., Zhang, L. J., Zhang, X. Y. and Li, T. 2018. Blueberry scorch virus detected on blueberry plants imported into？China. Plant Dis. 102: 1673.

상세보기

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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