Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.
A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.
Schnell
R J
RJ
USDA-ARS, National Germplasm Repository, Subtropical Horticulture Research Station, Miami, FL 33158, USA. schnell@usda-grin.gov
Olano
C T
CT
Kuhn
D N
DN
D001483:Base Sequence; D004279:DNA, Viral; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D014644:Genetic Variation; D019770:Lauraceae / Q000821:virology; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014772:Viroids / Q000235:genetics*
D004279:DNA, Viral; D005456:Fluorescent Dyes; D012367:RNA, Viral