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Development and Validation of a TaqMan qPCR Assay for the Detection of Citrus chlorotic dwarf associated virus
Avijit Roy - USDA-APHIS-PPQ-S&T-CPHST. Zhou Yan- Southwest University, Vessela Mavrodieva- USDA-APHIS-PPQ S&T CPHST, Charles Belanger- USDA-APHIS-PPQ-S&T-CPHST, John Hartung- USDA ARS MPPL, Changyong Zhou- Southwest University, Maria Saponari- CNR Sezione Di Bari, Mark Nakhla- USDA-APHIS-PPQ S

Citrus chlorotic dwarf disease (CCDD) is an emerging graft-transmissible disease first observed in the mid- 1980s in Turkey and in 2009 in China. CCDD is also transmitted by vector bayberry whitefly (Parabemisia myricae). CCDD symptoms include chlorotic flecking on young leaves, and crinkling, inverted cupping, variegation, chlorosis and dwarfing on mature leaves. A novel Geminivirus was discovered in association with CCDD in 2012 using high throughput sequencing, named Citrus chlorotic dwarf associated virus (CCDaV), and is listed as a quarantine pathogen in the United States. To prevent the introduction of the disease and to improve the existing CCDaV diagnostic methods, a triplex TaqMan qPCR assay was developed that targets the Coat Protein and RepA genes of the CCDaV genome along with the plant internal control COX gene. To develop a specific and sensitive molecular assay, CCDaV isolates from China, Italy and from the Exotic Pathogens Citrus Collection (EPCC) in BARC Beltsville MD, USA were obtained. The triplex TaqMan qPCR assay was optimized and validated by determining the selectivity, specificity, efficiency, repeatability and intermediate precision of the assay. No adverse effects were detected in the triplex assay when compared to the duplex assay for detecting one of the CCDaV targets and a COX plant DNA internal control gene. The described TaqMan qPCR assay can be used for quick and reliable detection of CCDaV from infected plant material.