ISHS Acta Horticulturae 583: I International Conference on Sweetpotato. Food and Health for the Future
DETECTION OF SWEETPOTATO CHLOROTIC STUNT CRINIVIRUS (SPCSV) BY NON-RADIOACTIVE NUCLEIC ACID SPOT HYBRIDIZATION (NASH) TECHNIQUE |
| Authors: | G. Müller, S. Fuentes, L.F. Salazar |
| Keywords: | SPVD, sweetpotato viruses |
| Abstract:
Sweetpotato viral disease (SPVD) caused by the synergistic interaction of sweetpotato feathery mottle potyvirus (SPFMV) and sweetpotato chlorotic stunt crinivirus (SPCSV) is difficult to recognize in infected plants of most cultivars. At present, specific monoclonal and polyclonal antibodies are available to detect both viral components of the disease. However, low concentration of SPCSV in plant tissues makes difficult its serological detection. A non-radioactive specific probe was developed to detect SPCSV using the published sequence of its coat protein gene. The non-radioactive probe was labeled by a random priming labeling technique with a fluoresceinated-nucleotide and assayed by Nucleic Acid Spot Hybridization (NASH). Different extraction buffers and modifications in the hybridization steps were assayed. Samples from field-grown sweetpotato showing typical SPVD-symptoms and samples without symptoms were collected and assayed by NCM-ELISA and NASH techniques. Comparative results showed that the non-radioactive NASH technique is slightly better than NCM-ELISA when samples are extracted with buffer 5X SSC and hybridized at 70°C for 4 h, using the hybridization buffer used routinely at CIP for Nick Translation NASH technique. This technique represents an important sensitive tool for the detection of SPCSV in "seed" multiplication approaches and also in programs devoted to development of SPVD-resistant genotypes. | |
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