The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests

Abstract

An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA coupled with amplicon detection via lateral flow assay (LFA). Primers labeled with fluorescein and biotin were designed for RT-RPA for effective recognition of the loop regions in the high-structured circular RNA of PSTVd. The labeled DNA amplicon was detected using lateral flow test strips consisting of a conjugate of gold nanoparticles with antibodies specific to fluorescein and streptavidin in the test zone. The RT-RPA-LFA detected 106 copies of in vitro transcribed PSTVd RNA in reaction or up to 1:107 diluted extracts of infected plant leaves. The assay took 30 min, including the RT-RPA stage and the LFA stage. The testing of healthy and infected potato samples showed full concordance between the developed RT-RPA-LFA and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the commercial kit. The obtained results proved the feasibility of using the developed assay to detect PSTVd from a natural source.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

“…Hence, a comparison of analytic characteristics of the developed test system is unfeasible. Based on the experience of previous works on the detection of other RNA plant viruses [ 9 , 11 , 42 , 43 ], we considered the developed test as appropriate for the detection of real AMV in infected plants.…”

“…The easiest way to obtain the labeled amplicon is by using forward and reverse RPA primers labeled at the 5′ ends with different tags (the scheme is presented in Figure 1 A). This approach is actively applied for the RPA-LFT detection of pathogens and species identification [ 8 , 9 , 10 , 11 ]. However, the probability of the cross-dimerization of the labeled RPA primers is high due to the typical RPA primers being rather long (28–35 nt), and there are no stages with the temperature denaturation of DNA.…”

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

“…First, all these tests were performed with the following commercial RPA kits: TwistDx nfo [27,84,[208][209][210][211], TwistDx Basic [212][213][214][215], TwistDx Exo [51], and Amplifier (Agdia) [27,92,[216][217][218]. The main criterion for the selection of a gene as a target for RPA was maximal specificity for the species of interest.…”

Antibodies as Biosensors’ Key Components: State-of-the-Art in Russia 2020–2021

“…The detection limit was 70 pM for a target of a length of 150 bp [87]. Murine mAbs specific to fluorescein (anti-FAM) (clone 2A3c, Bialexa, Russia) were used when developing a diagnostic kit based on the NALFIA principle to reveal the potato spindle tuber viroid [67]. In [81], a test for hepatitis B surface antigens was developed, in which the LFIA was improved by the electronic registration of superparamagnetic nanolabels.…”

“…Figure 4. Useful modifications of nanoparticles for improvement of the analyte detection described in this review: AuNPs[65][66][67][68][69][70][71][72][73], Superspherical AuNPs[74], Pt-nanoflower-type NPs[75], Au@PTNps[76], Au@Ag-PtNPs[77], quantum dots[78][79][80], magnetic beads[81].…”