Detection of TuMV by a toehold switch sensor coupled with NASBA amplification in Pseudostellaria heterophylla

Pseudostellaria heterophylla (P. heterophylla) is a perennial herb that has been used as a medicinal food for hundreds of years in China. Viral infections during the production of P. heterophylla severely reduce the yield and quality. Turnip mosaic virus (TuMV) is a common and highly variable viral pathogen in P. heterophylla plants. A high-efficiency diagnostic system is urgently needed to control and alleviate TuMV infection. However, the current detection methods still have various deficiencies that limit their field application. Here, a cell-free expression system relying on nucleic acid sequence-based amplification (NASBA) with toehold switch sensors and a visual reporter for color change was developed and introduced for TuMV detection in P. heterophylla. After designing and screening the approach, the selected sensitive sensor was able to detect 1 pM TuMV RNA fragments within 40 min, and the detection limit was less than 10 fM if the time was extended to 90 min. The sensor exhibited high specificity, with no cross-reactivity detected when tested against cucumber mosaic virus, another prevalent viral pathogen in P. heterophylla. In addition, in in-field samples, TuMV was successfully detected directly from both purified and crude RNA extracts in approximately 3 h. This cell-free synthetic biology tool is rapid, sensitive, specific and field-applicable and provides high-capacity and low-cost diagnostics for TuMV in P. heterophylla, as well as various viruses in herbs and other host plants.