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

ABSTRACTWe 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, instrument free assay for rapid nucleic acid screening in both hospitals and point-of-care settings.

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Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽

10.1371/journal.pone.0243319 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243319

Author(s):

Takeshi Hanami ◽

Tetsuya Tanabe ◽

Takuya Hanashi ◽

Mitsushiro Yamaguchi ◽

Hidetaka Nakata ◽

...

Keyword(s):

Nucleic Acid ◽

Single Molecule ◽

Detection System ◽

Signal To Noise Ratio ◽

High Sensitivity ◽

Nucleic Acid Detection ◽

Digital Measurement ◽

Excitation Light ◽

Target Nucleic Acid ◽

Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽

10.3390/bios11120479 ◽

2021 ◽

Vol 11 (12) ◽

pp. 479

Author(s):

Soumi Sukla ◽

Prasenjit Mondal ◽

Subhajit Biswas ◽

Surajit Ghosh

Keyword(s):

Nucleic Acid ◽

Dengue Virus ◽

Magnetic Beads ◽

Molecular Beacon ◽

Detection System ◽

Denv Infection ◽

Molecular Beacons ◽

Nucleic Acid Amplification ◽

Complementary Sequence ◽

Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

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Quantitative Analysis of Nucleic Acid Hybridization on Magnetic Particles and Quantum Dot-Based Probes

Sensors ◽

10.3390/s90705590 ◽

2009 ◽

Vol 9 (7) ◽

pp. 5590-5599 ◽

Cited By ~ 12

Author(s):

Sun Hee Lim ◽

Felix Bestvater ◽

Philippe Buchy ◽

Sek Mardy ◽

Alexey Dan Chin Yu

Keyword(s):

Quantitative Analysis ◽

Nucleic Acid ◽

Quantum Dot ◽

Magnetic Particles ◽

Nucleic Acid Hybridization

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Isolation of Monovalent Quantum Dot–Nucleic Acid Conjugates Using Magnetic Beads

Bioconjugate Chemistry ◽

10.1021/bc5002032 ◽

2014 ◽

Vol 25 (7) ◽

pp. 1342-1350 ◽

Cited By ~ 13

Author(s):

Uvaraj Uddayasankar ◽

Zhenfu Zhang ◽

Ravi T. Shergill ◽

Claudiu C. Gradinaru ◽

Ulrich J. Krull

Keyword(s):

Nucleic Acid ◽

Quantum Dot ◽

Magnetic Beads

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Strand displacement applied to assays with nucleic acid probes.

Clinical Chemistry ◽

10.1093/clinchem/32.9.1631 ◽

1986 ◽

Vol 32 (9) ◽

pp. 1631-1636 ◽

Cited By ~ 25

Author(s):

M S Ellwood ◽

M Collins ◽

E F Fritsch ◽

J I Williams ◽

S E Diamond ◽

...

Keyword(s):

Nucleic Acid ◽

Signal Sequence ◽

Target Sequence ◽

Strand Displacement ◽

Nonspecific Binding ◽

Nucleic Acid Probes ◽

Target Nucleic Acid ◽

Hybrid Complex ◽

Potential Applications ◽

Novel Method

Abstract This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.

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