DIG和碱性磷酸酶标记cDNA探针检测番木瓜PRSV的比较

利用地高辛(DIG)标记和碱性磷酸酶直接标记的cDNA探针核酸分子杂交及RT－PCR方法对番木瓜环斑病毒(PRSV)进行了检测。根据Genebank发表的PRSV中国Sm株系的外壳蛋白基因序列，设计特异引物，以美中红（Carica papaya L. cv. Meizhonghong ）带病样品的RNA为模板进行RT-PCR扩增反应,将其目的cDNA片段克隆到pGEM-T easy 质粒载体上，并测序。以重组质粒作模板，用PCR－DIG标记方法制备cDNA探针，另一种非放射性探针是经凝胶电泳分离、纯化cDNA片段，用碱性磷酸酶直接进行标记。结果表明，(1)美中红No.2序列与中国优势株系Sm的同源性为94.7%；(2) DIG标记的三种cDNA探针（861bp，455bp和215bp）对样品的总RNA检测结果是一致的, 且861bp的探针杂交斑点最为清晰，上述核酸杂交结果与RT－PCR检测结果相符, 核酸分子杂交检测的灵敏度和特异性能满足常规检测的需要；(3)碱性磷酸酶直接标记861bp的cDNA探针可进行PRSV的斑点杂交检测，而455bp的cDNA片段用碱性磷酸酶直接标记时，不能获得有效的杂交结果；(4)本试验还用DIG标记探针对叶脉进行了印迹杂交检测,结果与RT－PCR检测结果相符。

Comparison of Digoxgenin Labeling and AlkPhos Direct Labeling cDNA probes Detection Methods for Papaya Ringspot Virus（PRSV）

In this paper, we detected the Papaya Ringspot Virus (PRSV) on the papaya plants with nucleic acid hybridization by digoxgenin labeling; alkphos direct labeling cDNA probes and RT-PCR. The specific primers were designed according to published sequences of PRSV CP genes of Chinese Sm strain in Genebank to perform a RT-PCR, total RNA extracted from PRSV-infected samples，Carica papaya L.cv. Meizhonghong, cloned cDNA was linked to pGEM-T easy plasmid vector and sequence determinate. Recombinant plasmids were used as the template for labeling with PCR DIG Probe Synthesis Kit. Recovery the cDNA fragments by electrophoresis and labels cDNAs with AlkPhos Direct Labeling method. The result indicated that A. the sequence of the Meizhonghong’ No.2 isolate showed a 94.7 % identity with the Chinese PRSV-Sm genome. B. Blot hybridization obtained identical hybridization result when 861bp，455bp and 215bp probes labeled by DIG, and that 861bp probe lead to the clearest hybridization signal, above detected result were accord with that of RT-PCT, nucleic acid hybridization technique resulted in a sensitivity and special detection of PRSV in papaya, which is sufficient for routine diagnosis routine. C.The AlkPhos Direct Labeling 861bp probes could make for ideal blot hybridization result, and that the 455bp was invalidation. D. DIG labeling probe was performed in the vein tissue print hybridization in the determinations of PRSV.