1 research outputs found
CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever Virus
African swine fever
virus (ASFV) causes a highly contagious and
fatal disease affecting both domesticated and wild pigs. Substandard
therapies and inadequate vaccinations cause severe economic damages
from pig culling and removal of infected carcasses. Therefore, there
is an urgent need to develop a rapid point-of-use approach that assists
in avoiding the spread of ASFV and reducing economic loss. In this
study, we developed a colorimetric sensing platform based on dual
enzymatic amplification that combined the clustered regularly interspaced
short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a)
system and the enzyme urease for accurate and sensitive detection
of ASFV. The mechanism of the sensing platform involves a magnetic
bead-anchored urease-conjugated single-stranded oligodeoxynucleotide
(MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated
CRISPR/Cas12a. After magnetically separating the free urease, the
presence of virus can be confirmed by measuring the colorimetric change
in the solution. The advantage of this method is that it can detect
the presence of virus without undergoing a complex target gene duplication
process. The established method detected ASFV from three clinical
specimens collected from porcine clinical tissue samples. The proposed
platform is designed to provide an adequate, simple, robust, highly
sensitive and selective analytical technique for rapid zoonotic disease
diagnosis while eliminating the need for vast or specialized tools