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Open AccessArticle

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

1
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
2
A. G. Lorch Russian Potato Research Center, Kraskovo 140051, Russia
*
Author to whom correspondence should be addressed.
Plants 2020, 9(10), 1369; https://doi.org/10.3390/plants9101369
Received: 19 September 2020 / Revised: 9 October 2020 / Accepted: 12 October 2020 / Published: 15 October 2020
(This article belongs to the Special Issue Diagnosis and Control of Plant Viral Diseases)
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. View Full-Text
Keywords: isothermal amplification; lateral flow assay; potato spindle tuber viroid; recombinase polymerase amplification isothermal amplification; lateral flow assay; potato spindle tuber viroid; recombinase polymerase amplification
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MDPI and ACS Style

Ivanov, A.V.; Shmyglya, I.V.; Zherdev, A.V.; Dzantiev, B.B.; Safenkova, I.V. 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. Plants 2020, 9, 1369.

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