Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection

We have developed a novel detection system that couples clustered regularly interspaced short palindromic repeat-Cas recognition of target sequences, Cas-mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for simple detection of viral nucleic acid targets. After target recognition and Cas-mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic beads. A complementary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to uncleaved probes on the magnetic beads. After separating hybridized quantum dots, the collected supernatant is illuminated by a portable ultraviolet flashlight, and it provides a simple "Yes-or-No" nucleic acid detection answer. By using a DNA target matching part of the African swine fever virus, detection limits of ∼0.5 and ∼1.25 nM are achieved in buffer and porcine plasma, respectively. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple assay for nucleic acid screening in both hospitals and point-of-care settings.