A general RPA-CRISPR/Cas12a sensing platform for Brucella spp. detection in blood and milk samples

Abstract

Brucellosis is a disease that leads to long-term damage in human and livestock and is caused by Brucella spp., which is an important and dangerous pathogen. Accordingly, the rapid and accurate detection of Brucella spp. is critical for decreasing the rate of infection. Since the discovery of the trans-cleavage ability of Cas effector proteins, the CRISPR/Cas system has shown great potential in the development of next-generation biosensors for biomolecule detection. Here, we coupled the trans-cleavage activity of a CRISPR/Cas12a system with recombinase polymerase amplification (RPA) and used two analytical methods to develop a general sensing platform for Brucella spp. detection. The resultant RPA-CRISPR/Cas12a based fluorescent biosensor (F-CRISPR) and electrochemical biosensor (E-CRISPR) can detect as few as 2 copies/reaction of positive reference plasmid. Importantly, the present biosensors developed can determine four main Brucella strains and distinguish them from other non-target bacteria. Finally, these dual- biosensors based on RPA-CRISPR/Cas12a enable a rapid and accurate detection of Brucella spp. in milk (food) samples and blood (clinical) samples that is comparable to the traditional RT-PCR method, suggesting that this RPA-CRISPR/Cas12a sensing platform is a powerful tool in the early diagnosis of brucellosis.