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POSTERS: Pathogen detection, quantification and diagnosis

E-probe: A new diagnostic tool for detection of Dichorhaviruses associated with Citrus leprosis syndrome
Andres Espindola - Oklahoma State University. Avijit Roy- USDA-APHIS-PPQ-S&T-CPHST, Kitty Cardwell- Oklahoma State University, Vessela Mavrodieva- USDA-APHIS-PPQ S&T CPHST

Diagnosing of causal agents associated with Citrus Leprosis (CiL) syndrome has been difficult because of the large number of potential viruses. There are five cytoplasmic viruses that includes Cilevirus; Citrus leprosis virus (CiLV) -C, CiLV-C2, SJP strain of CiLV-C (CiLV-C-SJP), Hibiscus strain of CiLV-C2 (CiLV-C2H), Hibiscus green spot higrevirus -2 (HGSV-2), and five nuclear viruses including Dichorhavirus; CiLV-N, CiLV-N0, Citrus strain 1 and 2 of Orchid fleck virus (OFV-Cit1 and OFV-Cit2) and Citrus chlorotic spot virus (CiCSV). More viruses causing CiL are being discovered every year, making the development of a single RT-PCR based detection protocol a nearly impossible. Therefore, we created and validated a suite of diagnostic E-probes for Dichoraviruses (generic), Dichoraviruses infecting citrus, and near neighboring viruses on Orchid, Hibiscus, Clerodendrum and Coffee. There are 19 unique e-probe sets, each with in silico sensitivity analysis, analytical limit of detection and specificity curation. E-probes for all CiLV strains and near neighbors were selected to be 30nt long. Identifying (discriminatory) unique sequences per virus ranged from 2 – 20 e-probe sequences. Sensitivity was validated using simulated metagenomic databases containing the complete genome of navel orange and the generic Dichoraviruses for nuclear type species. The limit of detection in silico was 0.0034% relative abundance when using one million reads. Specificity of e-probes to discriminate different strains within a mixture of Dichoraviruses remains to be established.