Table 1.
Primer sequences of EMARA F7/R8 designed using Primaclade.
Primer thermodynamic features were calculated using Oligoanalyzer v. 3.1.
Fig 1.
RT-PCR optimization using cDNA of High Plains wheat mosaic virus (HPWMoV).
(A) Gradient PCR to determine optimum°Ta (45°C). (B) Assessment of Mg 2+ concentration and relative sensitivity to determine the optimal concentration (4 mM). Relative concentration of serially diluted cDNA: 1) 1 ng/μL. 2) 0.1 ng/μL. 3) 10 pg/μL. 4) 1 pg/μL. 5) 0.1 pg/μL. 6) 10 fg/μL. 7) 1 fg/μL.
Fig 2.
Multiple emaravirus detection assays.
(A) First assay, RT-qPCR-HRM normalized melting curve showing species-specific patterns, and corresponding endpoint RT-PCR products in agarose gel electrophoresis for European mountain ash ringspot-associated virus (EMARaV), fig mosaic virus (FMV) isolates dS, 1B and F3, rose rosette virus (RRV), High Plains wheat mosaic virus (HPWMoV), pigeonpea sterility mosaic virus 1 (PPSMV1), PPSMV2 and non-template control (NTC). Both PPSMV1 and PPSMV2 were not detected in this assay. (B) Second assay, RT-PCR detection of FMV isolates dS, 1B and F3, PPSMV1, PPSMV2, EMARaV, HPWMoV, RRV, and redbud yellow ringspot-associated virus (RYRSaV). Positive controls 1 and 2 are plasmids harboring diagnostic sequences for RRV and HPWMoV, respectively. The negative control is cDNA of healthy fig tissue. (C) Third assay, RT-qPCR-HRM, and endpoint RT-PCR detection of HPWMoV 07–961 (left) and RYRSaV (right). RT-PCR detection products in an agarose gel are featured left-center of each plot. Detection assays were performed at different time frames and in different laboratories.
Fig 3.
RT-qPCR-HRM fluorescence difference graphs calculated subtracting the normalized fluorescence graphs of each virus sample tested, starting from top left: High Plains wheat mosaic virus (HPWMoV), European mountain ash ringspot-associated virus (EMARaV), rose rosette virus (RRV), pigeonpea sterility mosaic virus 1 (PPSMV1), PPSMV2, fig mosaic virus (FMV) isolate 1B, FMV isolate F3, and FMV isolate dS.
Fig 4.
Sensitivity assays of positive controls (plasmids) containing a diagnostic sequence for High Plains wheat mosaic virus (HPWMoV), European mountain ash-ringspot associated virus (EMARaV), rose rosette virus (RRV) and fig mosaic virus (FMV) isolate 1B.
The LoD of primers EMARA F7/R8 was 1 fg (endpoint RT-PCR and RT-qPCR-HRM) for all the tested positive controls, except for EMARaV. Sensitivity for EMARaV was 10 fg in RT-PCR, and 1 fg for RT-qPCR-HRM. Plasmid concentrations are 1) 1 ng/μL. 2) 0.1 ng/μL. 3) 10 pg/μL. 4) 1 pg/μL. 5) 0.1 pg/μL. 6) 10 fg/μL. 7) 1 fg/μL. NTC: non-template control (DEPC-treated water).
Fig 5.
RT-qPCR calibration curve of positive control for High Plains wheat mosaic virus (HPWMoV) using primers EMARA F7/R8.
Plasmid concentrations are 1) 1 ng/μL. 2) 0.1 ng/μL 3) 10 pg/μL. 4) 1 pg/μL. 5) 0.1 pg/μL. 6) 10 fg/μL. 7) 1 fg/μL. NTC: non-template control (DEPC-treated water).
Fig 6.
Specificity assay showing inclusivity and exclusivity panels.
(A) Specificity assay using GoTAQ polymerase. Inclusivity panel: plasmids (positive controls) for High Plains wheat mosaic virus (HPWMoV), rose rosette virus (RRV) and European mountain ash ringspot-associated virus (EMARaV) were included. Exclusivity panel: cDNA of viruses co-infecting wheat and corn, wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV), viruses infecting roses, prunus necrotic ringspot virus (PNRSV), phylogenetically related orthotospoviruses, iris yellow spot virus (IYSV), groundnut ringspot virus (GRSV) mixed with tomato chlorotic spot virus (TCSV), tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), phylogenetically related tenuivirus, maize stripe virus (MSpV) and cDNA synthesized from healthy wheat-leaves RNA (negative control) were included. (B) Specificity assay using a hot-start polymerase. Inclusivity panel: plasmid for RRV. Exclusivity panel: cDNA for IYSV, INSV, TSWV, MSpV. On both assays, a non-template control (NTC) was included (DEPC-treated water).
Fig 7.
Phylogenetic analysis of protein inferred sequences derived from emaravirus nucleotide sequences amplified with primers EMARA F7/R8 in the first detection assay performed at Oklahoma State University.
Red diamond labels are reference emaravirus sequences from this study. The unrooted phylogenetic tree was generated using Maximum Likelihood method with 1000 bootstrap pseudo-replicates as branch support values. Numbers above nodes represent bootstrap values >50. The scale represents the number of substitutions per unit branch length. GenBank accession numbers for each used sequence are provided. Fig mosaic virus (FMV), PPSMV 1 and 2 = pigeonpea sterility mosaic virus 1 and 2, RRV = rose rosette virus, PiVB = Pistacia virus B, BLMaV = blackberry leaf mottle-associated virus, EMARaV = European mountain ash ringspot-associated virus, AcCRaV = Actinidia chlorotic ringspot-associated virus, RYRSaV = redbud yellow ringspot-associated virus, RLBV = raspberry leaf blotch emaravirus, TiRSaV = ti ringspot-associated virus, PVBV = blue palo verde broom virus, HPWMoV = High Plains wheat mosaic virus.
Table 2.
GC content, in-vitro, and in-silico melting temperatures of different tested emaraviruses.
In-vitro is the actual melting temperature (°Tm) after HRM. In-silico are the predicted°Tm calculated by uMeltSM. Number 1 are°Tm with σ as default, number 2 are the predicted°Tm adjusted with σ = 0.184407.