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.

中文翻译：

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快速检测高结构环状RNA的挑战：基于重组酶聚合酶扩增和侧向流动试验的马铃薯纺锤块茎类病毒测定

开发了一种检测马铃薯纺锤块茎类病毒（PSTVd）的方法，马铃薯是一种有害的植物病原体，会导致作物受损，从而导致马铃薯农业部门的经济损失。该测定基于PSTVd RNA的逆转录和重组酶聚合酶扩增（RT-RPA）以及通过侧向流动测定（LFA）进行的扩增子检测。设计有荧光素和生物素标记的引物用于RT-RPA，可有效识别PSTVd高结构环状RNA中的环区域。使用横向流动测试条检测标记的DNA扩增子，该测试条由金纳米颗粒与在测试区域中对荧光素和链霉亲和素具有特异性的抗体的缀合物组成。RT-RPA-LFA在反应中检测到10 ⁶份体外转录的PSTVd RNA拷贝或高达1:10⁷种被感染植物叶片的稀释提取物。该测定耗时30分钟，包括RT-RPA和LFA。健康和感染马铃薯样品的测试表明，开发的RT-RPA-LFA与定量逆转录聚合酶链反应（RT-qPCR）和市售试剂盒完全一致。获得的结果证明了使用开发的测定法从天然来源检测PSTVd的可行性。