TitleRecombinase Polymerase Amplification (RPA) for the Rapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus
Publication TypeJournal Article
Year of Publication2019
AuthorsDeShields, JB, Moroz, N, Braley, LE, Mora-Romero, GArlene, Tanaka, K
JournalAmerican Journal of Potato Research
Volume96
Start Page617
Issue6
Pagination617 - 624
Date Published2019/12/01
ISBN Number1874-9380
Abstract

The amplification of specific nucleic acid sequences with high specificity and sensitivity is an essential technique for pathogen detection. Recombinase polymerase amplification (RPA) is a rapid isothermal amplification method. Here, we demonstrate the end-point and real-time detection of Spongospora subterranea f. sp. subterranea (Sss) using RPA and potato mop-top virus (PMTV) using reverse transcription (RT)-RPA. Oligonucleotide primers were designed for amplification that target the internal transcribed spacer 1 (ITS1) region and the coat protein readthrough (CP-RT) domain for the detection of Sss and PMTV, respectively. Our data showed that real-time RPA can detect 100 of Sss sporosori per gram of soil, while real-time RT-RPA could detect in ~1 ng total RNA of the PMTV-infected tuber. For Sss detection, the R2 value for real-time RPA and real-time PCR was 98.0% by linear regression analysis in the concentration range of 100–34,000 sporosori per gram of soil. The developed RPA assay here may provide a useful alternative tool for the rapid, simple and reliable detection of Sss and PMTV in diagnostic laboratories and in-field testing.

URLhttps://link.springer.com/article/10.1007/s12230-019-09750-7#citeas
DOI10.1007/s12230-019-09750-7
Short TitleRapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus