应用RT-PCR、斑点杂交法和SDS-PAGE检测水稻黑条矮缩病毒

Detection of rice black-streaked dwarf Fijivirus by RT-PCR, dot-blot hybridization and SDS-PAGE

用RT PCR技术、PCR标记的探针点杂交和SDS PAGE检测了生产上严重危害玉米和水稻的水稻黑条矮缩病毒(RBSDV)。由RT PCR扩增的RBSDV第 7片段第 92 1～ 14 11碱基作探针 ,用PCR法DIG标记后点杂交 ,可以从 10 0ng玉米病叶中检测到RBSDV ,灵敏度是RT PCR的 1/10 ;10 %SDS PAGE只能检测到从 1g玉米病叶中提取的病毒dsRNA ,但对江苏玉米田间分离的不同的RBSDV样品电泳发现 ,该病毒基因组dsRNA有差异 。

The purpose of this study was evaluation of RT PCR technique,dot blot hybridization with PCR generated probe and SDS PAGE for the detection of rice black streaked dwarf Fijivirus(RBSDV).The cDNA probe labeled with digoxigenin by PCR,complementary to the region between 921 and 1 411 nucleotides of segment 7 of RBSDV from Jiangsu,was used.In dot blot hybridization the probe could detect the viral RNA from 100 ng diseased maize leaves,which is ten times less sensitive than RT PCR.10% SDS PAGE only could detect the viral dsRNA extracted from 1 g diseased maize leaves,but this technique was found to be simple and effective to study the genomic diversity of RBSDV isolated in fields.