title: Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.
abstract: Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Kwon
authors.affiliation: Department of Applied Biology, Kangwon National University, Chunchun, Republic of Korea.
authors.forename: Jin Sung
authors.initials: JS
authors.lastname: Hong
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea.
authors.forename: Min Jea
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea.
authors.forename: Sun Hee
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea.
authors.forename: Byeong Eun
authors.initials: BE
authors.lastname: Min
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea.
authors.forename: Eun Gyeong
authors.initials: EG
authors.lastname: Song
authors.affiliation: Department of Life Science, Sahmyook University, Seoul, Republic of Korea.
authors.forename: Hyun Hee
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Plant Virus Genbank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women's University, Seoul, Republic of Korea. Electronic address: ryu@swu.ac.kr.
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Ryu
mesh_terms: D028464:Cucurbita / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D014777:Virus Diseases / Q000821:virology*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Cucurbit; Diagnosis; Multiplex; Polydeoxyinosine; Quarantine; RT-PCR