An artificial positive control for routine detection of rose rosette virus and Phyllocoptes fructiphilus that fit most primers for PCR, LAMP and RPA based assays

Abstract

Rose (Rosa spp.) is a very important ornamental shrub cultivated worldwide and of value for the pharmaceutical industry. The plant is broadly susceptible to pathogens, including viruses. Rose rosette virus (RRV; virus species Emaravirus rosae) causes multiple symptoms typically rosettes, ultimately leading to death. The virus transmission is by grafting and a wind-dispersed eriophyoid mite, Phyllocoptes fructiphilus, which survives in winter-dormant plants. Due to extensive globalization RRV is a threat for the European rose, landscape, nursery and tourism industries. The most common and reliable method used for RRV detection is RT-PCR. Positive control is indispensable for PCR reliability and can be difficult to obtain for emerging or highly contagious pathogens and are subject to BSL-2 quarantine. A synthetic artificial positive control (APC) using custom DNA inserts of sense and anti-sense primers was designed de novo and inserted in a circular plasmid vector to create a positive control for use with most RRV reported primers and eriophyoid mites. This study describes a functional demonstration and development of a rapid, consistent, adaptable and cost-effective alternative to infected true-tissue positive control for detection of RRV. The inserted RRV primers are for end point and quantitative RT-PCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP), recombinase-polymerase amplification (RPA), broad detection of emaravirus and the eriophyoid mite vector Phyllocoptes fructiphilus. The APC-RRV and RRV infected rose (leaf tissue) were tested side to side. Results demonstrated APC-RRV is a safe, cloneable and reliable approach subjected to quality control with application in quarantine surveillance and routine diagnostics of RRV.