Sensitive Detection of Avocado Sunblotch Viroid (ASBVd) Using Fluidigm Real Time and Digital PCR Assays

Avocados are the most important agricultural crop in South Florida. Recently, a new avocado disease, laurel wilt, has been observed in Miami-Dade County. The SHRS houses an extensive avocado germplasm collection which is being threatened by laurel wilt. We are in the process of backing up our collection at the USDA station in Hilo, HI. Unfortunately, some of our germplasm accessions are also infected with ASBVd, a single-stranded circular viroid that causes sunblotch disease. Because Hawaii has an avocado industry and no known ASBVd, we have had to develop an extremely sensitive detection method for the viroid to prevent shipping infected budwood from our germplasm collection. We isolate RNA from the plant, preamplify with primers that recognize the whole 243 nt molecule and then use a TaqMan assay on the Fluidigm BioMark™ System (for real time PCR) or EP1™ System (for digital PCR) to detect the presence of ASBVd. With a known infected tree as the positive control, we have been able to reliably detect ASBVd at a 107 dilution of the original sample, using the 192x24 IFC on the BioMark™ System and assaying twelve replicates of each dilution. We have successfully identified infected accessions that were not detected with our previous PCR assay of fragment analysis on the ABI 3730.