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| Comparison of Digoxgenin Labeling and AlkPhos Direct Labeling cDNA probes Detection Methods for Papaya Ringspot Virus（PRSV） |
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Abstract 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.
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Received: 04 July 2006
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