Ivanov Aleksandr V, Shmyglya Irina V, Zherdev Anatoly V, Dzantiev Boris B, Safenkova Irina V
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
A. G. Lorch Russian Potato Research Center, Kraskovo 140051, Russia.
Plants (Basel). 2020 Oct 15;9(10):1369. doi: 10.3390/plants9101369.
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 copies of in vitro transcribed PSTVd RNA in reaction or up to 1:10 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.
开发了一种检测马铃薯纺锤块茎类病毒（PSTVd）的检测方法，PSTVd是一种危险的植物病原体，会导致作物受损，给马铃薯农业部门造成经济损失。该检测方法基于PSTVd RNA的逆转录和重组酶聚合酶扩增（RT-RPA），并通过侧向流动分析（LFA）检测扩增子。设计了用荧光素和生物素标记的引物用于RT-RPA，以有效识别PSTVd高度结构化环状RNA中的环区域。使用由金纳米颗粒与检测区中针对荧光素和链霉亲和素的抗体的缀合物组成的侧向流动测试条检测标记的DNA扩增子。RT-RPA-LFA在反应中检测到10个体外转录的PSTVd RNA拷贝，或在感染植物叶片的提取物稀释至1:10时仍能检测到。该检测方法耗时30分钟，包括RT-RPA阶段和LFA阶段。对健康和感染马铃薯样品的检测表明，所开发的RT-RPA-LFA与定量逆转录聚合酶链反应（RT-qPCR）和商业试剂盒之间完全一致。所得结果证明了使用所开发的检测方法从天然来源检测PSTVd的可行性。
