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

• Published in: Annals of applied biology Vol. 183; no. 1; pp. 67 - 79
• Main Authors: Ruschel, Rafaela Gomes, Taylor, Mason, Ochoa‐Corona, Francisco M., Amirudeen, Abdul Kader Jailani, Druciarek, Tobiasz, Paret, Mathews
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.07.2023; Wiley Subscription Services, Inc
• Subjects: Amplification; APC; artificial positive control; cost effectiveness; death; diagnostic techniques; diagnostics; DNA; emaravirus; Globalization; Inserts; landscapes; leaves; Mites; monitoring; ornamental woody plants; Pathogens; Pharmaceutical industry; Phyllocoptes fructiphilus; Plant tissues; plasmid vectors; Polymerase chain reaction; Primers; Quality control; Quarantine; Recombinase; Reverse transcription; reverse transcription loop-mediated isothermal amplification; rose rosette; Rose rosette emaravirus; Rosette; Tourism; virus transmission; Viruses
• ISSN: 0003-4746; 1744-7348
• DOI: 10.1111/aab.12834

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. The application of the artificial positive control (APC) concept allows the development and design of individual control fragments for a large number of diverse and specified targets. If implemented on large scale, APCs will find a wide range of applications in diagnostics and microbiology. The synthetic insert of APC‐RRV consists of tandems of selected RRV oligonucleotide primer and probe sequences of 16 reported genomic targets of RRV, a conserved emaravirus motif, and mite barcoding primers. 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.