Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification

Abstract

Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples.

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum

“…RPA is a relatively new isothermal amplification method that can amplify target DNA to detectable levels in less time and at lower temperatures than that of other isothermal amplification techniques [28–30]. In addition, RPA is tolerant to impure samples [31] and its reagents are available in a lyophilized form that can be transported without requiring cold chain storage. The above advantages make it particularly suitable for field diagnosis.…”

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

“…All samples were analysed separately by RT-PCR with the Dz actin F and Dz actin R primers designed in this study from a D. zingiberensis actin gene sequence. This enabled confirmation that a negative result was because of the lack of viral RNA rather than the presence of reaction inhibitors in the template, avoiding false-negative results which commonly have a negative impact in routine YMV diagnosis (Mekuria et al, 2014;Mumford and Seal, 1997).…”

“…These methods offer simple and rapid detection systems, with high specificity and sensitivity and are suitable for field diagnosis and poorly equipped laboratories (Amer et al, 2013;Boyle et al, 2013;Escadafal et al, 2014). RPA tests have been described recently for the diagnosis of important plant viruses such as Little cherry virus 2 (Mekuria et al, 2014) and Plum pox virus . One reason this sequence-specific isothermal nucleic acid amplification method has emerged as a novel technology in molecular diagnosis, is that it offers quick and sensitive detection without the need for thermal cycling as required for PCR (Zanoli and Spoto, 2013).…”

Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

“…The recombinase polymerase amplification (RPA) is a very attractive alternative that overcomes all of the drawbacks of the other isothermal approaches42. An increasing number of reports detailing different formats of RPA are appearing and the technique gives true promise for application at the point-of-need via sensors or lateral flow formats43444546474849505152535455.…”