Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a Technology for Rapid and Highly Sensitive Detection of Heterodera avenae and Heterodera filipjevi.

Abstract

The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10-4 single second-stage juvenile (J2), 10-5 single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10-1 single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.