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.

Aleksandr V Ivanov,Irina V Shmyglya,Boris B Dzantiev,Irina V Safenkova,Anatoly V Zherdev

doi:10.3390/plants9101369

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.

Highlights

The potato spindle tuber viroid (PSTVd) is the most primitive potato plant pathogen that causes perceptible crop damage [1]
The first stage of developing the recombinase polymerase amplification (RPA)-lateral flow assay (LFA) for PSTVd involved designing a set of labeled primers and testing them with model PSTVd RNA
The first stage of developing the RPA-LFA for PSTVd involved designing a set of labeled primers andF2 testing them with model PSTVd RNA

Summary

Introduction

The potato spindle tuber viroid (PSTVd) is the most primitive potato plant pathogen that causes perceptible crop damage [1]. PSTVd can infect different hosts belonging to the Solanaceae family. As a part of the Pospiviroidae family, PSTVd is a circular, rod-like, structured RNA molecule with a central conservative region and variable “rod” terminals. There are more than 200 PSTVd variants with an average length of 359 nt. The secondary structure of PSTVd is non-complete double stranded RNA (dsRNA), consisting of loops and non-canonical base-pairing [2]. The RNA molecules can be divided into five domains: terminal right, terminal left, central conservative regions, pathogenicity-related

Methods

Results

Conclusion