Discussion
The LAMP/Cas12a assay accurately detected RS phylotype Ⅰ in 14 test strains, and showed low detection limit (2.0 × 100 copies). RS in tomato stem tissue and soil samples from two field sites with suspected BW infection was identified accurately, suggesting potential application of LAMP/Cas12a assay as point-of-care test (POCT). The overall detection process took less than 2 h and did not require professional lab equipment. Our findings, taken together, indicate that LAMP/Cas12a assay can be developed as an effective, inexpensive technique for field detection and monitoring of RS.
Methods
We describe here a novel RS detection assay based on combination of loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a. crRNA1, with high trans-cleavage activity targeting hrpB gene, was selected out of four candidate crRNAs. Two visual detection techniques, involving naked-eye observation of fluorescence and lateral flow strips, were tested and displayed high sensitivity and strong specificity.