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

Abstract

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.

Recent advances in CRISPR‐based systems for the detection of foodborne pathogens

“…Bao et al. (2020) also enhanced detection sensitivity, by incorporating a probe system that uses quantum dots (Qdots). Target recognition cleaves the ssDNA probe molecule, enabling the Qdot reporter probe to emit a fluorescent signal.…”

“…The properties of these noble-metal nanomaterials maximized the difference in intensity between the positive and negative signal, resulting in enhanced sensitivity without amplification. Bao et al (2020) also enhanced detection sensitivity, by incorporating a probe system that uses quantum dots (Qdots). Target recognition cleaves the ssDNA probe molecule, enabling the Qdot reporter probe to emit a fluorescent signal.…”

Advanced Nanomaterials for Preparedness Against (Re‐)Emerging Viral Diseases

“…Recently, Du et al have developed a CRISPR-Cas12a detection system using QDs as a reporter to detect ASFV-related DNA targets. [127] Magnetic beads were used together to sequester uncleaved probe DNA linked QDs after target-specific CRISPR cleavage, reducing the background signal. The fluorescence of the QDs remaining in the solution was then recorded to estimate the target concentration.…”

CRISPR/Cas technology as a promising weapon to combat viral infections

“…When LbCas12a RNP and its target are previously added to the biotin‐labeled probes, the target‐activated RNP cleaves the biotin probe and the Qdot‐probes cannot bind the MBs, so the solution remains colored after magnetic MB separation. [ 115 ] This is a highly portable system due to the naked‐eye readout. Its low sensitivity could be improved by coupling it with prior sample amplification.…”