CRISPR-Cas12a Test Strip (CRISPR/CAST) Package: Poverty Family Pasture Health Patron Saint for Livestocks Free from Brucella Infection

Brucellosis is a common zoonotic disease caused by Brucella , which causes enormous economic loss and public burden to the epidemic areas. Earlier and precise diagnosis and timely culling of infected animals are crucial to prevent the infection of Brucella and the spread of the disease. In recent years, RNA-guided CRISPR/Cas12a nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid on-site screening, especially on the remote family pasture. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated protein 12a (Cas12a), the CRISPR/Cas12a system combined with recombinase polymerase amplification(RPA), and lateral flow read-out. The CRISPR/CAST package can complete the assay of Brucella nucleic acid within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl, and no antigen cross-reacting against Yersinia enterocolitica O:9 , Escherichia coli O157 , Salmonella enterica serovar Urbana O:30 , and Francisella tularensis . The serum samples of 398 sheep and 100 cattle were tested by CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR and higher than the Rose Bengal Test (RBT, 19 sheep, and 5 cattle were serum positive). CRISPR/CAST package can accurately detect the infected livestock’s Brucella DNA and accomplish within 30 min, which has the advantages of simple, fast, high sensitivity, and strong specificity, with no window period. Besides, the package needs no expensive equipment, standard laboratory, or professional operators. It is an effective tool for field screening and earlier, rapid diagnosis of Brucella infection. A package is an efficient tool for epidemic prevention and control.