Poster: Diseases of Plants: Disease Detection & Diagnosis
Rose rosette virus detection using loop-mediated isothermal amplification (LAMP)
A. SALAZAR AGUIRRE (1), S. Molina Cárdenas (1), F. Ochoa-Corona (2), J. Olson (2) (1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.
Rose rosette virus (RRV) is an Emaravirus transmitted by Phyllocoptes fructiphilus strongly associated with Rose rosette disease (RRD). Visual diagnosis maybe confusing and specific, sensitive, easy to use with visual detection methods are required. LAMP is an isothermal amplification that combines these criteria. Alignment of 20 RRV P4 (RNA 4) accessions allowed primer design seeking broad detection of reported isolates. Optimal amplification was obtained with Bst 2.0 WarmStart® DNA polymerase (0.32 u), outer primers F3 and B3 (0.2 µM), internal primers FIP and BIP (0.8 µM) and MgSO4 (4 mM) at 66.5°C for 1 hour. Bovine serum albumin (BSA) (4 mg/mL) and polyvinylpyrrolidone (PVP) (1%) were added as isothermal amplification enhancers. The detection limit was 1 pg/µL with plasmid carrying the targeted sequence. Products were visualized by electrophoresis. The visual detection limit of plasmid in colorimetric reactions using Hydroxynaphthol blue (HNB) (120 µM) without BSA and PVP was 0.01 ng/µL. No cross-reactions with cDNA from ten frequently rose co-infecting or related viruses (HPV, MSV, INSV, TSWV, GRSV, ApMV, ArMV, PNRSV, TRSV and TMV) was detected. Healthy tissue and non-template controls were included in all reactions. RRV-LAMP tested successfully with 25 tissue samples of symptomatic RRD roses from Oklahoma. The method has potential applications in biosecurity, microbial forensics and nursery virus-free monitoring of germplasm.