Apr 12, 2020

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

BioRxiv : the Preprint Server for Biology
Mengdi BaoKe Du

Abstract

We have developed a novel detection system which couples CRISPR-Cas recognition of target sequences, Cas mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for instrument-free 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 particles. A complimentary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to un-cleaved probes on the magnetic beads. After separation of hybridized from unhybridized quantum dot conjugates by magnetic sequestration, the signal is measured fluorometrically to provide a signal proportional to the cleaved probes and therefore the amount of target nucleic acid. To demonstrate the power of this assay, a 250 bp DNA target sequence matching a portion of the African swine fever virus (ASFV) genome is used and a detection limit of ~0.5 nM is achieved without target amplification using a simple portable ultraviolet flashlight. 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, instrumen...Continue Reading

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Mentioned in this Paper

Single-Cell Analysis
Crossed Reflex
Genes
Neoplastic Cell
Gene Expression
RNA, Small Cytoplasmic
Cell Type
Subclone
Analysis
Transcriptome

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