Poster: Diseases of Plants: Disease Detection & Diagnosis
Virus detection of Tobamovirus with wide spectrum degenerate oligonucleotides by TD RT-PCR High Resolution Melting
J. GARCIA SUAREZ (1), S. Dobhal (1), F. Ochoa-Corona (1) (1) Oklahoma State University, U.S.A.
Tobamoviruses cause notable plant diseases worldwide in Solanaceae, Cucurbitaceae, orchids and some are reported to be waterborne. Molecular tools exist for detection of this genus, however, a detection method combining genus detection and species discrimination is needed. Eleven reference positive controls from the DMSZ repository (Germany) and AGDIA (USA), were studied. Complete Tobamovirus genomes were aligned and a single antisense primer was designed to target the RNA dependent RNA polymerase (RdRp). This antisense primer co-reacted with five sense primers for selective subgroup detection by RT-PCR and HRM discrimination of eleven species within five subgroups. A protocol for detection and discrimination based on Touchdown Reverse transcription-Polymerase Chain Reaction (TD RT-PCR) coupled to High Resolution Melting (HRM) was developed. Species specific dF/dT Tm curves ranged 75-85°C and were generated for each primer groups as follow: I (BpEMV, PMMoV, TMV, ToMV), II (PaMMV, TMGMV), III (SFBV, RMV), IV (ORSV, CGMMV) and V (SHMV). The method allows the incorporation of new groups of tobamoviruses to the assay by designing additional group specific sense primers. The resulting 11 diagnostic sequences showed 98-100% identity regarding the targeted viruses. HRM facilitates a reliable discrimination of species specific profiles for each virus species. The method is amenable to microbial forensics diagnosis and biosecurity applications.