pathogenic bacteria

Sensors and Actuators B Chemical

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. • We firstly introduce a general RPA-CRISPR/Cas12a sensing platform into fluorescent and electrochemical dual-signal output. • We firstly established an isothermal, rapid, specific and ultrasensitive RPA-CRISPR/Cas12a sensing platform for Brucella spp. detection. • This platform enabled rapid and accurate detection of Brucella spp. in milk and blood samples, which was better than RT-PCR. • This general sensing platform with a dual-signal output detection could be generalised to detect other pathogens.

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