Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus

This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/μL and 1 fg/μL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/μL and 0.1 pg/μL using hydroxy naphthol blue (HNB, 120 μM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.

LAMP results can be visualized by the naked eye using either a pre-reaction and pH-sensitive dye (Hydroxynaphthol Blue) or a post-reaction fluorescent dye (PicoGreen or SYBR Green I) [19,20].
Among, the best management practices once the RRD occurs is to remove symptomatic plants including the root ball seeking to minimize secondary inoculum and the spreading of viruliferous mites to healthy plants, which in time causes economic losses [1]. Accurate, sensitive, and field transferable detection of RRV is needed for early detection of RRV in foundation blocks, susceptible breeding lines, and biosecurity purposes. This study hypothesizes the technological need can be fulfilled by exploring the development of a LAMP method while investigating chemistries and dyes available for naked eye visualization.

Material and methods
A flow chart showing the RRV genes targeted, and the sequence of methods and chemistries explored is shown in Fig 1. Tulsa rose garden, Oklahoma. Nine lyophilized reference-positive control of viruses commonly infecting roses were from Agdia, Inc (Agdia, Elkhart, IN) and used for specificity assays. These virus species are impatiens necrotic spot virus (INSV), High Plains wheat mosaic virus (formerly High Plain virus) (HPWMoV), arabis mosaic virus (ArMV), maize stripe virus (MSpV), tomato spotted wilt virus (TSWV), apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), tomato ringspot virus (ToRSV), and tobacco mosaic virus (TMV). Healthy tissue of Rosa multiflora was used as a negative control.

LAMP primer design
Twenty-two RRV nucleoprotein gene sequences (RNA3) were retrieved from the NCBI Gen-Bank. The accession numbers of the analyzed sequences are HQ891892.1-HQ891913.1. Twenty sequences of the RRV movement protein gene (RNA4) were retrieved from the NCBI GenBank database. The accession numbers of analyzed sequences are HQ891870.1-HQ891889.1. The last date of accession was November 17th, 2019.
The NCBI accessions selected for LAMP primers design were RRV isolates collected in Arkansas, Mississippi, Missouri, Alabama, Tennessee, Iowa, and Oklahoma.
Sequences of LAMP oligonucleotides primers were designed using the web interface application Primer Explorer (Eiken Chemical Co., Ltd.) (http://primerexplorer.jp/e/). A set of LAMP primers was selected (Table 1) following parameters described in the Primer Explorer Manual (S1 Table). The specificity of the LAMP primers was tested in silico using BLASTn [21]. LAMP primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA).

RNA extraction
RNA extractions were made from both fresh or lyophilized rose tissue and commercially available virus reference controls (Agdia, Elkhart, IN). For fresh tissue, approximately 100 mg of leaves, petals, bark, or roots were loaded in 2 mL microcentrifuge tubes and pulverized using liquid nitrogen and mini-pestels. Total RNA was extracted using the RNeasy plant mini kit (Qiagen Inc., Valencia, CA) following the instructions of the manufacturer. For Agdia lyophilized reference positive controls, 450 μL of RLT buffer (from the Qiagen RNeasy plant mini kit) was directly aliquoted into the positive control vial, vortexed for 30s, and the total RNA extracted following the manufacturer's instructions.

Virus direct antigen-capture method
Viral RNA was obtained from leaves, petals, bark from young stems, and roots using direct antigen-capture or direct trapping in plastic as described by Babu et al. [18]. Briefly, rose tissue was macerated in 1 mL of phosphate buffer saline (1X PBS) (VWR, Radnor, PA), 0.05% Tween 20, and pH 7.4. Fifty microliters of sap extract were aliquoted into a sterile PCR tube (0.5 ml polypropylene), avoiding air bubbles. The sap was incubated on ice for 2 min, then, the PCR tubes were rinsed twice with 50 μL of 1X PBS-T buffer. To release the viral RNA from the plastic-surface captured virion, 30 μL of nuclease-free water with RNAsin (Promega, Madison, WI) (100 U/mL, final concentration) was added and incubated at 95˚C for 1 min. The supernatant was directly used in reverse transcription reactions.

cDNA synthesis
Four microliters of extracted total RNA obtained either with an RNA extraction kit or the direct antigen-capture method were used. The protocol was performed in two steps. First, denaturation followed by reverse transcription. LAMP RRVP3-B3 or RRVP4-B3 primers (5 μM) were used rather than random hexamer primers, and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Promega, Madison, WI), following the manufacturer's instructions. cDNA synthesis was performed at 37˚C for 90 min.

RT-PCR and cloning of RRV-P3 and RRV-P4 fragments
RT-PCR products were amplified with both LAMP external primers RRVF3-B3 or RRVF4-B4. The PCR reaction mix used was: 10 μL GoTaq Green Master Mix (Promega, Madison, WI), 1 μL RRVP3/F3 and RRVP3/B3 or RRVP4/F3 and RRVP4/B3 primers (5 μM), 3 μL of cDNA template, 1.6 μL of BSA (bovine serum albumin, 50 mg/mL; Ambion, Austin, TX), 2 μL 10% PVP40 (polyvinylpyrrolidone, Sigma-Aldrich, St. Louis, MO), and 1.4 μL nuclease-free water (Promega, Madison, WI). The final volume of the PCR reaction was 20 μL. The RT-PCR reaction was performed in a thermal cycler (Biometra, Goettingen, Germany) and cycling parameters were as follows: initial denaturation of 94˚C for 5 min, 40 cycles of denaturation at 94˚C for 20 s, annealing at 56˚C for 20 s, extension at 72˚C for 20 s, and a final extension at 72˚C for 5 min. The amplified products were analyzed by 1X TAE-2% agarose gel electrophoresis (Tris-acetate-EDTA) and stained with SYBR Safe (Invitrogen, Waltham, MA) according to the manufacturer. The 100bp DNA Ladder (Promega, Madison, WI) was added in all the gel electrophoresis analyses. The RRV-P3 and RRV-P4 RT-PCR products were excised and purified from the agarose gel in two stages. First, by QuantumPrep Freeze'N Squeeze Spin Columns (Bio-Rad, Hercules, CA), followed by a second purification with High Pure PCR Product Purification Kit (Roche, Germany). The TOPO TA cloning kit (Invitrogen, Waltham, MA) was used for cloning the purified PCR fragments segments of RRV-P3 and RRV-P4 genes. The TOPO TA kit was used according to the manufacturer's instructions. Briefly, the previous purified PCR products were ligated into the commercial pCR´4-TOPO plasmid and transformed into Escherichia coli cells (TOP10, Mach1™-T1R, DH5α™-T1R cells) following the manufacturer's instructions. Plasmids were sequenced by Sanger sequencing and stored at -20˚C until use.

RT-LAMP optimization
The purified plasmids carrying the RT-PCR inserts of RRV-P3 and RRV-P4 were used for optimization purposes. The optimal temperature was assessed from a gradient of temperature assay from 60˚C to 72˚C and the optimal MgSO4 concentration was determined by testing nine different concentrations from 2mM to 10mM.
RT-LAMP was performed in a dry bath incubator (GeneMate/Bioexpress, Kaysville, UT). RT-LAMP reactions for RRV-P3 were incubated for 1 h at 64˚C and 1h at 66.5˚C for RRV-P4. The polymerase deactivation was performed at 80˚C for 10 min, at the end of the reaction. The amplified products were analyzed by electrophoresis in 2% agarose gel, 1X TAE buffer, and stained with SYBR Safe (Invitrogen, Waltham, MA). In order to visualize the RT-LAMP products, two colorimetric reactions were studied and optimized. The pre-colorimetric reaction was assessed using HNB, which was included in the RT-LAMP mix reaction. On the other hand, the post-colorimetric reaction was performed using 3 μL of freshly prepared 10-fold dilution of SYBR Green I dye (Invitrogen, Waltham, MA), which was added after the RT-LAMP incubation period (Fig 1). Cresol red (16 mM) and Malachite green (0.2%) (Sigma-Aldrich, St Louis, MO) were also tested to assess their pre-colorimetric reaction using Bst 2.0 WarmStart 1 DNA polymerase.

Limit of detection and specificity assays
LoD assays were performed using ten-fold serial dilutions from 1 ng/μL to 1 fg/μL of plasmids RRV-P3 and RRV-P4. The plasmid concentration was quantified using a Nanodrop 2000 (Thermo Scientific, Waltham, MA). One microliter of each dilution was used as a template for RT-LAMP.
For specificity, the LAMP primers were RNA extracted from nine lyophilized reference virus controls (Agdia, Elkhart, IN) listed above in the section 'source of viruses'. Plasmid RRV-P3 (positive control), cDNA and RNA from healthy rose tissue (negative control), and nuclease-free water (non-template control) were included in both LoDs and specificity assays. The results of these assays were analyzed in 2% agarose electrophoresis in 1X TAE buffer, colorimetric reaction, and RT-qLAMP.

Screening of field samples
RRV-P4 RT-LAMP was tested with 38 field samples including symptomatic and non-symptomatic rose samples collected at the Oklahoma State University RRV resistance rose varietal plot by PDIDL. RRV-P3 RT-LAMP was tested with 30 samples of rose tissue collected at the Tulsa rose garden. The RT-LAMP reaction conditions were according to the optimized parameters tested and described above. Endpoint RT-PCR was performed as a reference and confirmatory assay using the RRV primers and cycling conditions described by Dobhal et al. [11].

LAMP primer design
The primers were selected after analysis of the thermodynamic parameters described by the Primer Explorer manufacturer for the selection of LAMP primers (S1 Table). LAMP primer sets for RRV-P3 and RRV-P4 aligned with the consensus regions of twenty-two RRV-P3 sequences and twenty RRV-P4 sequences ( Table 1). The result showed 100% identity with 100% query coverage for both groups of gene sequences of RRV (P3 and P4), after the alignment analysis of outer LAMP primers RRV-P3/F3, RRV-P3/B3, RRV-P4/F3, and RRV-P4/B3. Matches with other emaraviruses and virus species were not detected.

RT-LAMP optimization
The RT-LAMP optimization (primer concentration, temperature gradient, and MgSO 4 concentration) was carried out with plasmid harboring fragments of RRV-P3 and RRV-P4 genes amplified by endpoint RT-PCR with outer LAMP primers RRVP3-F3, RRVP3-B3, RRVP4-F3, and RRVP4-B3. The sequencing output of the two cloned plasmids had 99% identity to the RNA3 and RNA4 of RRV isolates in the NCBI database.

Primer concentration.
Since LAMP primer sets perform at different concentrations, three concentrations of the inner LAMP primers RRV-FIP and RRV-BIP (0.8, 1.6, and 2 μM) were tested with a single concentration of the outer primers RRV-F3 and RRV-B3 (0.2 μM). The best concentration of outer primers was 0.2 μM and 0.8 μM for inner primers, which produced the best visible and distinct band pattern indicative of a positive LAMP reaction amplification (Fig 2). We optimized the concentration for both sets of primers (RRV-P4 and RRV-P3) and the results were consistently the same for the two sets. Therefore, we only showed in Fig 1. the RRV-P3 results.

RT-LAMP performance in temperature gradient.
Primer set RRV-P3 amplified the expected product within 63 to 68˚C (Fig 3A). Similarly, LAMP performed well in a broad range of temperatures amplifying the expected RRV-P4 gene diagnostic product from 60 to 66˚C (Fig 3B). The annealing temperature ranged from five and six degrees Celsius respectively. The amplification temperatures selected for LAMP reactions were 64˚C for RRV-P3 and 66.5˚C for RRV-P4 for one hour. No product amplification was detected with the nontemplate control using the two LAMP primer sets.

Concentration of magnesium sulfate.
The RRV-P3 and RRV-P4 diagnostic products were amplified within six MgSO4 concentrations: 2, 3, 4, 5, 6, and 7 mM (Fig 4A) using Bst 2.0 DNA polymerase and the pre-reaction and pH-sensitive dye HNB. The 4 mM MgSO4 concentration allowed the amplification and visual discrimination of RRV targets compared with non-template reactions. The same MgSO 4 optimization results were obtained using both RRV-P3 and RRV-P4. The MgSO 4 RRV-P3 RT-LAMP optimization is show in Fig 4. Negative reactions were purple and positive reactions were light blue (Fig 4B). MgSO 4 concentrations 6-9 mM are not optimal ( Fig 4A) and cannot be colorimetrically distinguishable (Fig 4B). Cresol red and Malachite green did not react in the colorimetric reaction using Bst 2.0 WarmStart DNA polymerase and no change of color was observed (data not shown).

PLOS ONE
Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus RT-LAMP specificity assay using HNB using cDNA of nine reference control viruses. Tube N, is a non-template control (NTC, water), tube P is RRV-P3 plasmid, tube 1 is INSV, tube 2 is HPWMoV (formerly High plain virus), tube 3 is ArMV, tube 4 is MSpV, tube 5 is TSWV, tube 6 is ApMV, tube 7 is PNRSV, tube 8 is ToRSV, tube 9 TMV (full virus names were provided in the main text for the reference viruses), tube R, healthy rose tissue. (C) Colorimetric RRV-P3 RT-LAMP specificity assay using SYBR Green I. tube 1 is INSV, tube 2 is HPWMoV, tube 3 is ArMV, tube 4 is MSpV, tube 5 is TSWV, tube 6 is ApMV, tube 7, is PNRSV, tube 8 is ToRSV, tube 9 TMV (full virus names were provided in the main text for the reference viruses), tube R is healthy rose tissue, tube S is RRV symptomatic rose tissue, tube P is RRV-P3 plasmid, and tube N is a non-template control (NTC, water). The negative controls did no amplify or generated a colorimetric reaction with healthy rose tissue, and the NTC (water) as expected. In general, detection results using RRV-P3 RT-qLAMP with GspSSD2.0 and RT-PCR agreed with the LoD reported and the varietal results were consistent across Tables 3 and 4. The method for direct antigen-capture was consistently trapping RRV directly in plastic from different tissue sources of roses. The addition of SYBR Green I post RT-LAMP reaction generated color change toward fluorescent green in positive amplification, while no change of color or steady-orange was consistent in all negative reactions (no amplification), healthy rose. Amplification and colorimetric reaction (SYBR Green I) were obtained with the RVV-P3 plasmid.

Discussion
This study explores, describes, and demonstrates the development of specific, sensitive, and relatively easy-to-use RRV RT-LAMPs. The study used two sets of primers for RT-LAMP amplification of RRV-P3 and RRV-P4 genes, which were tested with Bst 2.0 and GspSSD2.0 DNA polymerases. These methods have the potential to be field deployable. RT-LAMP was also tested using SYBR green I and HNB to generate qualitative colorimetric reactions, and an RT-qLAMP was tested.
The viral nucleic acids, in this case, RNA, required for RT-LAMP reactions is commonly extracted from plant tissue using commercially available kits. However, RNA isolated from roses may carry phenolic compounds, starch, other carbohydrates, pigments, and other plant residues in high levels. These compounds inhibit the targeted cDNA and/or DNA

PLOS ONE
amplification because they interact with the viral nucleic acids and the reaction mix proteins causing oxidation and degradation of the RNA [22]. All of which decreases the quality of the samples and causes inconsistent results and concerns about whether all RRV isolates are detectable. Dobhal et al. [23], enhanced RRV RT-PCR detection by adding DNA AFs such as BSA and PVP minimizing the inhibitory effect of putative rose components. However, it was observed the colorimetric RT-LAMP-HNB reaction did not develop a visually distinguishable (sharp) change of color between positive and negative reactions when BSA and PVP were added to the reaction, yet obtained amplification is detectable by gel electrophoresis (data not shown). The development of color in colorimetric RT-LAMP-HNB reactions relies on a  Screaming red neon (2) Stem --

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Champlain ( change in pH occurring during the reaction, and the incorporation of AFs such as BSA and PVP may be interfering with the acidification of the reaction [24]. The direct antigen-capture method reported by Babu et al. [18] allowed rapid and consistent trapping of RRV virions directly on plastic (inner PCR tube surface). The method washes the rose tissue impurities and improves the quality of RNA used in RT-LAMP reactions and it substantially reduces the sample processing time. The method also reduces viral RNA extraction time for LAMP, RPA, and/or RT-PCR to less than 5 min. Simple RNA extraction methods combined with RT-LAMP have been performed to reduce the detection time. The wooden toothpick was tested to collect Fig mosaic virus (FMV) for direct transfer to RT-LAMP; this method can detect the virus in 30 min; however, sampling should be taken from a symptomatic plant region; otherwise, detection could be a negative result [25]. In contrast, the direct antigen capture method uses a mix of plant samples including asymptomatic samples that can still be detected by RT-LAMP. In summary, the direct antigen-capture method applied to RT-LAMP allowed the RNA reverse transcription and amplification of targeted DNA with no need for BSA and PVP enhancers. Furthermore, the direct antigen-capture did not interfere with pre-reaction pH-sensitive dye (hydroxy naphthol blue) and post-reaction dye (SYBR Green I) in colorimetric RT-LAMP reactions.
The LAMP primers were design based on RNA3 and RNA4 of the RRV genome because these segments were highly conserved among the isolates available in the NCBI database. In addition, comparing RNA1 (RNA dependent RNA polymerase -RdRp) and RNA3 (nucleocapsid), RNA3 could be encoding high levels of nucleocapsid protein; therefore, the RNA3 segment is a good candidate for targeting the RRV [7,26]. The use of only four primers per genekit facilitated RT-LAMP optimization, assay design, and application of optimized RT-LAMP parameters i.e. optimal outer and inner primer ratios, reaction temperature, and best  concentration of magnesium sulfate, which successfully favored the amplification of the expected RRV-P3 and RRV-P4 targets at 64˚C and 66.5˚C respectively (Figs 2, 3 and 4). Having RT-LAMP performing in a broad range of reaction temperatures reduces errors due to temperature variability that might occur associated with equipment calibration. The selected MgSO4 concentration (4mM) allowed steady amplification of both RRV-P3 and RRV-P4 targets. The optimal concentration of the inner and outer LAMP primers is 0.8μM and 0.2 μM respectively, equivalent to a 4:1 primer ratio. The described Bst 2.0 driven RT-LAMP amplification can be translated to the field or nursery testing if using the fluorescent (SYBR green I).
The pH-sensitive dyes (HNB) are less recommended. An advantage of LAMP colorimetric results is that can be judged either at daylight or UV light. A critical difference to consider if pursuing field-testing is that SYBR Green I has to be added at the end of the RT-LAMP reaction, while HNB must be added to the mix before the RT-LAMP reaction starts (Fig 1). To mitigate the risk of contamination while opening the tubes a top layer of mineral oil was added to all reactions. SYBR green I allowed clear visual discrimination between infected and healthy samples compared to HNB (Figs 5B, 6B and 6C). The visual LoD of SYBR green I and HNB are 0.01ng/μL and 0.1pg/Ll, respectively. The LoD between the two LAMP chemistries studied was different. Bst 2.0-based RT-LAMP detected RRV to 1pg/μL while GspSSD2.0 RT-qLAMP allows detection to1fg/μL. The RT-qLAMP assay from this study and the RRV RT-PCR reported by Dobhal et al. [11] have equivalent LoDs (1fg/μL) (Fig 8).
The two RRV-RT-LAMP sets of primers did not cross-react with eight reference control virus commonly co-infecting rose viruses, one taxonomically related emaravirus to RRV, and healthy rose tissue, which confirmed the predicted specificity pairwise analysis results obtained in-silico using BLASTn. In terms of specificity, no difference between LAMP primer sets RRV-P3 gene (viral coat protein) and RRV-P4 gene (movement protein) was detected (Fig 9).
The RT-qLAMP assay is relatively less time-consuming because was combined with the direct antigen-capture or direct trapping in plastic which takes 10-15 minutes for a batch of 1-15 samples. The one-step GspSSD2.0 RT-qLAMP reaction takes approximately 1 hour and uses RNA directly as a template while the two-step RT-PCR reaction time takes circa 2 hours and uses cDNA as a template. cDNA requires an additional 45 min to 1 hour. In this research, GspSSD2.0 RT-qLAMP was performed with a thermocycler (RotorGene 6000), but fluorescence recordings can be also performed using a simpler fluorescent reader that allows on-site testing of doubtful symptomatic rose plants. The side-by-side comparison of RT-LAMP and RT-PCR [11] showed inconsistent detection of RRV by RRV-P4 RT-LAMP with Bst 2.0-polymerase if compared to RT-PCR (Table 2). Twelve samples tested negative out of 38 RT-PCR positives. The discrepancy between the two methods (13 samples) is due to their differences in LoD, 1pg/μl for RT-LAMP, and 1fg/μl for RT-PCR [11]. Improvement in LAMP primer design and adding loop primers (LF and LB) would improve the LoD of the method. A second sideby-side comparison between RRV-P3 RT-qLAMP using GspSSD2.0 and the colorimetric RRV-P3 RT-LAMP using Bst 2.0 combined with SYBR Green was performed with 33 rose samples (Table 3). Twelve samples tested positive using the two methods and five samples tested positive only with RRV-P3 RT-qLAMP GspSSD2.0. This discrepancy is due to the difference in LoD between RT-qLAMP (1fg/μl) and RT-LAMP using BST 2.0 with SYBR Green I (0.1pg/μl), Figs 5 and 6. Another group of 17 samples tested positive using RRV-P3 RT-qLAMP with GspSSD2.0, and thirteen samples tested positive using RRV-P3 RT-LAMP with Bst 2.0 combined with SYBR Green I, other 16 samples tested negative with these two methods. Testing leaves, bark, and roots from different varieties demonstrated the uneven distribution of RRV in rose plants which is detailed in Table 3. Regarding the uneven distribution of the virus, sampling should be performed from different plant parts to have a higher possibility to detect RRV. However, further research is needed to determine the best sampling procedure for RRV detection.
A third side-by-side comparison of RRV-P3 RT-qLAMP GspSSD2.0, colorimetric RRV-P3 RT-LAMP Bst 2.0 with SYBR Green I, and RT-PCR was made targeting seventeen rose samples collected at the RRV resistance rose varietal plot located at the Tulsa rose garden (Table 4). This experiment also shows discrepancies in detection among the three methods. The GspSSD2.0 detected 2 RRV positives out of the 17 samples that were not detected by the colorimetric Bst 2.0 DNA Polymerase plus SYBR Green I and RT-PCR. These results are consistent with the low LoD of Bst 2.0 reported and the varietal results were consistent across Tables 3 and 4.
The uneven distribution of RRV in plants may be the cause of RRV passing not adverted by RT-PCR. In general, RRV was detected from leaves, petals, stems (bark), and roots. Consistent amplification of RRV was obtained from leaves, however, inconsistencies using RT-PCR point toward the uneven distribution of RRV in their hosts and the need for a statistically based sampling method. In

Conclusion
This study explored RT-LAMP with two DNA polymerases, RT-qLAMP with GspSSD2.0 which can be confirmed by gel electrophoresis, and colorimetric RT-LAMP with Bst 2.0. Four different dyes were tested, but visible colorimetric reactions were obtained only when RT-LAMP Bst 2.0 was combined with SYBR I or HNB. The last is less recommended since the change of color is low contrast. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR, takes a shorter time since it works with RNA directly and one-step reaction, however, does not support colorimetry. RT-LAMP with Bst 2.0. does not have an LoD as low as RT-PCR, but it supports colorimetry. Colorimetric RT-LAMP Bst2.0 also takes additional reaction time since requires a reverse-transcription step. In general, the tested isothermal LAMPs have the potential for field application, monitoring virus-free germplasm in nurseries, selection of RRV-resistant germplasm, biosecurity surveillance at points of entry, and microbial forensics.
Supporting information S1