Rapid identification of quarantine invasive Solanum elaeagnifolium by real-time, isothermal recombinase polymerase amplification assay

The rapid identification and assay of quarantine Solanum weeds is currently an enormous challenge due to the similar morphology of the seed and seedling of Solanaceae. Herein, we report the development of an easy-to-implement strategy to identify Solanum elaeagnifolium by utilizing recombinase polymerase amplification (RPA) technology. The strategy can be performed in less than one hour without harsh reaction conditions or expensive detection equipment, demonstrating a huge potential for fast on site identification. To monitor the amplification process, an oligonucleotide probe flanked by a dT-fluorophore and a corresponding dT-quencher group with homology to the target was designed to indicate the real-time fluorescence intensity during amplification. The increase of the fluorescence signal can be observed within 10 min at 39 °C. The limit of detection (LOD) of the real-time RPA assay of S. elaeagnifolium was estimated to be 2.55 pg genomic DNA, and the limit of quantification (LOQ) was estimated to be 25.5 pg genomic DNA. The rapid nature of the RPA assay and its low energy requirements compared to other amplification technologies suggested that RPA-based assays could be used for field screening of quarantine weeds and species.