Detection of Cucumber green mottle mosaic virus in Bottle Gourd Seeds by RT-PCR

Lee, Sook Kyung; Song, Wan Yeob; Kim, Hyung Moo

Research in Plant Disease 2004;10(1):53-57.
Published online March 30, 2004.

RT-PCR에 의한 박 종자의 오이녹반모자이크바이러스 검정

이숙경, 송완엽, 김형무

Detection of Cucumber green mottle mosaic virus in Bottle Gourd Seeds by RT-PCR

Sook Kyung Lee, Wan Yeob Song, Hyung Moo Kim

Abstract

Cucumber green mottle mosaic virus (CGMMV) was a major pathogen of watermelon and had affected seriously to watermelon production in Korea. Rapid and sensitive detection method of CGMMV associated with bottle gourd (Lagenaria siceraria) seeds was developed by using RT-PCR in this study. A pair of primer, Wmf1 and Wmr1, specific for CGMMV was designed from coat protein gene sequences of CGMMV-W and used for amplifying 420 bp product in RT-PCR. To simplify the virus extraction procedure and reduce an inhibitor from the extract for the RT-PCR, some methods using ethanol precipitation, double filtration, polyethylene glycol precipitation and phenol/chloroform/ isoamyl alcohol extraction procedure were compared and the phenol/chloroform/isoamyl alcohol extraction procedure was selected by its enhanced sensitivity. This detection method using the selected extraction step and the primers for RT-PCR could reliably detect an infected level of one CGMMV-infested seed in 1,000 seeds. This rapid and sensitive RT-PCR assay provides a useful tool for the specific detection of CGMMV in bottle gourd seed samples containing high levels of background inhibitors.

Key Words: Bottle gourd seed, CGMMV, phenol/chloroform/isoamyl alcohol extraction procedure, RT-PCR