Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.
Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field.
Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Kannondai, Tsukuba-City, Ibaraki 305-0856, Japan. Electronic address: mokuda@affrc.go.jp.
Mitsuru
M
Okuda
National Institute for Environmental Studies, Onogawa, Tsukuba-City, Ibaraki 305-8506, Japan.
Shiori
S
Okuda
The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. Electronic address: topcrop@agri.kagoshima-u.ac.jp.
Hisashi
H
Iwai
D000818:Animals; D029383:Crinivirus / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D006430:Hemiptera / Q000821:virology*; D007303:Insect Vectors; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013696:Temperature
D017931:DNA Primers
Cucurbit chlorotic yellows virus; Sticky trap; Virus detection; Whitefly