Dot-blot Hybridization For Detection Of Cucurbit Viruses By Digoxigenin-Labelled CDNA Probes

To provide a good alternative assay in seed health test, epidemiological and transgenic research, dot-blot hybridization was developed to detect five cucurbit viruses -Zuccini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Papaya ringspot virus- watermelon strain (PRSV-W) and Squash mosaic virus (SqMV) .Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers. The probes were applied in dot-blot hybridization to detect ZYMV,WMV,CMV,PRSV-W and SqMV in crude extraction of infected leaves. And three SqMV probes of different lengths (0.55, 1.6, 2.7 kb) were designed to research probe length's effect on hybridization. To detect the five virus in 18 different infected leaves in different areas and years and 15 different infected zucchini leaves in Zhengzhou in 2007 with the assay.The sensitivity for detection of the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W and SqMV was down to 1: 160, 1: 160, 1: 320, 1: 160, 1: 320 respectively. The 3 SqMV probes of different lengths cauesed no differences on hybridization effect. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stablities, sensitivities, specificity and reproducibilities. Five viruses could be detected from the infected leaves with the assay.