Abstract
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.
Highlights
Roses are affected by a wide variety of fungi, bacteria, viruses, nematodes, and phytoplasmas, which cause growth reduction, defoliation, and plant death [1]
Samples were sourced by the Plant and Insect Disease Diagnostic Laboratory (PDIDL), Oklahoma State University (OSU), and were collected from two rose rosette virus (RRV) resistance rose-varietal plots located in Perkins and the
It was observed the colorimetric RT-Loop-Mediated Isothermal Amplification (LAMP)- Hydroxy Naphthol Blue (HNB) reaction did not develop a visually distinguishable 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
Summary
Roses are affected by a wide variety of fungi, bacteria, viruses, nematodes, and phytoplasmas, which cause growth reduction, defoliation, and plant death [1]. In most cases, these pathogens attack flowers or leaf tissue causing economic losses to landscapers, gardeners, public gardens, and nurseries [1]. Rose rosette disease (RRD) is one of the most devastating diseases of roses. This disease was reported in 1940 in Manitoba, Canada, and it has since spread to roses in Kansas, Oklahoma, Missouri, and Arkansas [3]. The rose garden never recovered, all roses got lost and the garden lots have been used to assess rose varietal resistance to RRD
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