SUMMARY
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/Cas 12a T est
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 Brucellanucleic acid within 30 min under isothermal temperature conditions, with
a sensitivity of 10 copies/μl, and no antigen cross-reacting againstYersinia 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 wereBrucella 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 ofBrucella infection. A package is an efficient tool for epidemic
prevention and control.
KEYWORDS: Brucella infection, CRISPR-Cas12a, RPA, Test
strip, CRISPR/CAST package