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
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...
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MDPI AG
2020-10-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/9/10/1369 |
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| author | Aleksandr V. Ivanov Irina V. Shmyglya Anatoly V. Zherdev Boris B. Dzantiev Irina V. Safenkova |
| author_facet | Aleksandr V. Ivanov Irina V. Shmyglya Anatoly V. Zherdev Boris B. Dzantiev Irina V. Safenkova |
| author_sort | Aleksandr V. Ivanov |
| collection | DOAJ |
| description | 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 10<sup>6</sup> copies of in vitro transcribed PSTVd RNA in reaction or up to 1:10<sup>7</sup> 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. |
| first_indexed | 2024-03-10T15:35:15Z |
| format | Article |
| id | doaj.art-266d93fab5fa468cb37f66b75168fb4f |
| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-10T15:35:15Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-266d93fab5fa468cb37f66b75168fb4f2023-11-20T17:16:03ZengMDPI AGPlants2223-77472020-10-01910136910.3390/plants9101369The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow TestsAleksandr V. Ivanov0Irina V. Shmyglya1Anatoly V. Zherdev2Boris B. Dzantiev3Irina V. Safenkova4A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, RussiaA. G. Lorch Russian Potato Research Center, Kraskovo 140051, RussiaA.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, RussiaA.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, RussiaA.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, RussiaAn 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 10<sup>6</sup> copies of in vitro transcribed PSTVd RNA in reaction or up to 1:10<sup>7</sup> 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.https://www.mdpi.com/2223-7747/9/10/1369isothermal amplificationlateral flow assaypotato spindle tuber viroidrecombinase polymerase amplification |
| spellingShingle | Aleksandr V. Ivanov Irina V. Shmyglya Anatoly V. Zherdev Boris B. Dzantiev Irina V. Safenkova 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 isothermal amplification lateral flow assay potato spindle tuber viroid recombinase polymerase amplification |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | challenge for rapid detection of high structured circular rna assay of potato spindle tuber viroid based on recombinase polymerase amplification and lateral flow tests |
| topic | isothermal amplification lateral flow assay potato spindle tuber viroid recombinase polymerase amplification |
| url | https://www.mdpi.com/2223-7747/9/10/1369 |
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