title: Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination.
abstract: In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2U Bst DNA polymerase and 4mM MgSO4 for 1h at 60-62 °C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea; The Institute of Life Science and Technology, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Eui-Joon
authors.initials: EJ
authors.lastname: Kil
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Sunhoo
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea; Crop Protection Division, National Academy of Agricultural Science, Rural Development Administration, Wanju 565-851, Republic of Korea.
authors.forename: Ye-Ji
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea; Plant Quarantine Technology Center, Animal and Plant Quarantine Agency, Suwon 443-400, Republic of Korea.
authors.forename: Eun-Ha
authors.initials: EH
authors.lastname: Kang
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Minji
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Cho
authors.affiliation: Crop Protection Division, National Academy of Agricultural Science, Rural Development Administration, Wanju 565-851, Republic of Korea.
authors.forename: Mi-Kyeong
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Institute of Plant Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Kyeong-Yeoll
authors.initials: KY
authors.lastname: Lee
authors.affiliation: Plant Quarantine Technology Center, Animal and Plant Quarantine Agency, Suwon 443-400, Republic of Korea.
authors.forename: Noh-Youl
authors.initials: NY
authors.lastname: Heo
authors.affiliation: Crop Protection Division, National Academy of Agricultural Science, Rural Development Administration, Wanju 565-851, Republic of Korea.
authors.forename: Hong-Soo
authors.initials: HS
authors.lastname: Choi
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Electronic address: cell4u@skku.edu.
authors.forename: Sukchan
authors.initials: S
authors.lastname: Lee
mesh_terms: D029383:Crinivirus / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005189:False Positive Reactions; D007723:Korea; D018551:Solanum lycopersicum / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D012680:Sensitivity and Specificity; D013696:Temperature; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D051981:Uracil-DNA Glycosidase
keywords: Carry-over contamination; False-positive reaction; Loop-mediated isothermal amplification (LAMP); Tomato chlorosis virus (ToCV); Uracil-DNA glycosylase (UDG)