Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis

A specific reverse transcription-polymerase chain reaction (RT-PCR) protoco l has been developed for routine detection of avocado sunblotch viroid (ASB Vd). Modifications in this diagnostic technique were made to enable fluores cent detection and variant identification using automated capillary electro phoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP ) analysis. Sixteen sequence variants characterized in a previous study wer e analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant diff erences were detected between data obtained from the two ABI 310 Genetic An alyzers indicating that an internal control must be run concurrently with t he samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provide d support for the visual differences in SSCP patterns. The use of SSCP in t he ASBVd assay is easily accomplished and gives an estimate of the number o f variants in crude samples extracted from infected avocado plants.