Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.

Staphylococcus aureus (S. aureus) is an important opportunistic human and animal pathogen that can cause a wide diversity of infections. Due to its environmental health risks, it is crucial to establish a time-saving, high-throughput, and highly sensitive technique for water quality surveillance. In this study, we developed a novel method to detect S. aureus in the water environment based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a. This method utilizes isothermal amplification of nucleic acids and the trans-cleavage activity of the CRISPR/Cas12a system to generate fluorescence signals with a single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter and a naked-eye detected lateral flow assay (LFA). Our RPA-CRISPR/Cas12a detection system can reduce the detection time to 35 min and enhance the high-throughput detection threshold to ≥5 copies of pathogen DNA, which is more sensitive than that of reported. Moreover, in the lower reaches of the Jialing River in Chongqing, China, 10 water samples from the mainstream and 7 ones from tributaries were successfully monitored S. aureus for less than 35 min using RPA-CRISPR/Cas12a detection system. Taken together, a novel high-throughput RPA-CRISPR detection was established and firstly applied for sensitively monitoring S. aureus in the natural water environment.