生物素标记cDNA探针杂交检测梨树上3种潜隐病毒研究

 本研究合成了苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的生物素标记cDNA探针,对斑点杂交和免疫印迹杂交检测这3种病毒的效果进行了分析。以含ACLSV和ASGV克隆片段的大肠杆菌菌液为样品时,斑点杂交检测灵敏度分别为80 cfu/μL和50 cfu/μL。以离体培养砂梨植株为材料,采用斑点杂交法检测砂梨离体培养植株粗提液中的3种病毒,均产生强的杂交信号,且特异性好。比较斑点杂交和ELISA检测病毒含量相对较低的热处理再生植株中ASGV和ACLSV,结果表明斑点杂交具有较高的灵敏度。组织印迹杂交检测砂梨离体植株ASGV和ACLSV的结果显示,这2种病毒在离体植株的各部位均有分布,自基部至茎尖各部位印迹均产生很强的杂交信号。

Detection of three latent viruses in pear by hybridization with biotinylated cDNA probes

Biotinylated cDNA probes specific to Apple stem grooving virus(ASGV), Apple cholortotic leaf spot virus(ACLSV) and Apple stem pitting virus(ASPV) were prepared from cloned fragments. Efficiency of dot-blot and tissue printing hybridization for detection of these three viruses was evaluated. The detection sensitivities of dot-blot hybridization for E. coli containing cloned fragments were 80 cfu/μL and 50 cfu/μL for ACLSV and ASGV respectively. The prepared biotinylated cDNA probes could specifically recognize(ASGV,) ACLSV and ASPV in crude extract of in vitro-cultured pear plants and give strong hybridization signals. Comparison of detection results for ASGV and ACLSV in regenerated in vitro-cultured pear plants from thermotherapy revealed a higher sensitivity of dot-blot hybridization than ELISA. Tissue printing hybridization was also successfully used for detection of ASGV and ACLSV in in vitro-cultured pears. The results showed that these two viruses distributed in the whole plants.