地高辛标记探针检测5种葫芦科作物病毒的斑点杂交方法

Dot-blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

【目的】探索5种葫芦科作物病毒即小西葫芦黄花叶病毒(Zuccini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番木瓜环斑病毒西瓜株系(Papaya ringspot viruswatermelon strain,PRSV-W)和南瓜花叶病毒(Squash mosaic virus,SqMV)的斑点杂交检测技术,为葫芦科作物种子带毒的快速准确鉴定、流行病学研究和转基因检测等提供技术和方法。【方法】以构建的重组质粒为模板,用PCR方法合成了相应的地高辛标记的cDNA探针。通过斑点杂交检测西葫芦病叶汁液来考察探针的灵敏性和特异性。同时设计了3种不同长度的SqMV探针(0.55、1.6、2.7kb)对杂交效果进行了研究。【结果】ZYMV、WMV、CMV、PRSV-W和SqMV的5种探针检测各自侵染西葫芦病汁液的稀释低限分别为1﹕160、1﹕160、1﹕320、1﹕160、1﹕320,而每种探针与健康西葫芦和其它4种病毒的反应均为阴性。不同长度的SqMV探针杂交结果相同。【结论】用PCR方法合成的地高辛标记的探针,能够直接在植物组织中将5种病毒检测出来,具有较好的灵敏性、特异性和重复性。探针的长度对杂交的灵敏性和特异性没有影响。

【Objective】 To provide a good alternative assay in seed health test,epidemiological and transgenic research, dot-blot hybridization was developed to detect five viruses infecting cucurbitaceous crops-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).【Method】Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with 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 the effect of hybridization.【Result】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 three SqMV probes of different lengths showed no differences.【Conclusion】The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of five viruses infecting cucurbitaceous crops with good stability, sensitivity, specificity and reproducibility.