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

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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A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus

Sensors ◽

10.3390/s20133728 ◽

2020 ◽

Vol 20 (13) ◽

pp. 3728

Author(s):

Ching-Chou Wu ◽

Hao-Yu Yen ◽

Lu-Ting Lai ◽

Guey-Chuen Perng ◽

Cheng-Rei Lee ◽

...

Keyword(s):

Nucleic Acid ◽

Dengue Virus ◽

Electrochemical Impedance ◽

Denv Infection ◽

Viral Disease ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Great Promise ◽

Label Free ◽

Hybridization Efficiency

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

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Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03309-4 ◽

2021 ◽

Author(s):

Noemi Bellassai ◽

Roberta D’Agata ◽

Giuseppe Spoto

Keyword(s):

Nucleic Acid ◽

Structural Properties ◽

Molecular Beacon ◽

Dna Origami ◽

Molecular Beacons ◽

Conformational Polymorphism ◽

Functional Systems ◽

Similarities And Differences ◽

Nucleic Acid Structures ◽

Nucleic Acid Sequences

AbstractNucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

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Virus concentration using polyethyleneimine-conjugated magnetic beads for improving the sensitivity of nucleic acid amplification tests

Journal of Virological Methods ◽

10.1016/j.jviromet.2003.08.002 ◽

2003 ◽

Vol 114 (1) ◽

pp. 11-19 ◽

Cited By ~ 33

Author(s):

K Satoh

Keyword(s):

Nucleic Acid ◽

Magnetic Beads ◽

Nucleic Acid Amplification ◽

Virus Concentration ◽

Nucleic Acid Amplification Tests

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Integrating nucleic acid sequence-based amplification and microlensing for high-sensitivity self-reporting detection

The Analyst ◽

10.1039/d0an01231a ◽

2020 ◽

Vol 145 (23) ◽

pp. 7528-7533

Author(s):

Feiyue Teng ◽

Xinpei Wu ◽

Tao Hong ◽

Gary B. Munk ◽

Matthew Libera

Keyword(s):

Electron Beam ◽

Nucleic Acid ◽

Solid Phase ◽

High Sensitivity ◽

Molecular Beacons ◽

Solution Phase ◽

Nucleic Acid Amplification ◽

Nucleic Acid Sequence ◽

Viral Detection

We use electron-beam patterned functional microgels to integrate self-reporting molecular beacons, dielectric microlenses, and solid-phase and/or solution-phase nucleic acid amplification in a viral-detection microarray model.

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Comparison of the Nucleic Acid Amplification System NASBA® and Agar Isolation for Detection of Pathogenic Campylobacters in Naturally Contaminated Poultry

Journal of Food Protection ◽

10.4315/0362-028x-59.7.683 ◽

1996 ◽

Vol 59 (7) ◽

pp. 683-687 ◽

Cited By ~ 14

Author(s):

M. UYTTENDAELE ◽

R. SCHUKKINK ◽

B. VAN GEMEN ◽

J. DEBEVERE

Keyword(s):

Nucleic Acid ◽

False Positive ◽

Detection System ◽

False Negative ◽

Isolation Procedure ◽

Nucleic Acid Amplification ◽

True Nature ◽

Selective Enrichment ◽

Amplification System ◽

Positive Results

A total of 160 poultry products were examined for the presence of pathogenic campylobacters using the traditional agar isolation method and the nucleic acid amplification system NASBA®, both after a 24-h selective enrichment. Pathogenic campylobacters could be isolated from 92 of 160 (57.5%) samples using agar isolation, among which 79 (49.37%) were identified as Campylobacter jejuni, six (3.75%) as C. coli, five (3.12%) as C. lari, and two (1.25%) as unclassified. The NASBA® assay provides a specific and sensitive method for detection of these campylobacters. A total of 149 samples (93.12%) gave similar results for both the traditional isolation procedure on modified Campylobacter charcoal desoxycholate agar and the NASBA® enzyme-linked gel assay detection system. Two false-negative results were obtained with the agar isolation procedure. Nine false-positive results were reported when the NASBA® system was used. However, the high sensitivity of the NASBA® method and indications that in some cases the traditional isolation procedure failed (abundance of a contaminating noncampylobacter bacteria which grew on the Campylobacter selective media) raises doubt about the true nature of these false-positive results. The NASBA® detection assay offers a rapid and useful analytical method when screening for the presence of pathogenic campylobacters. The complete procedure, including 24 h of selective enrichment, required 32 h.

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Novel Benzoxazole Inhibitor of Dengue Virus Replication That Targets the NS3 Helicase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02251-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1902-1912 ◽

Cited By ~ 56

Author(s):

Chelsea M. Byrd ◽

Douglas W. Grosenbach ◽

Aklile Berhanu ◽

Dongcheng Dai ◽

Kevin F. Jones ◽

...

Keyword(s):

Dengue Virus ◽

Molecular Beacon ◽

Single Point ◽

Denv Infection ◽

Geographic Region ◽

Single Point Mutation ◽

Helicase Domain ◽

Viral Pathogen ◽

Dengue Disease

ABSTRACTDengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replicationin vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

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Detection of potential microbial antigens by immuno-PCR (PCR-amplified immunoassay)

Journal of Medical Microbiology ◽

10.1099/jmm.0.070318-0 ◽

2014 ◽

Vol 63 (5) ◽

pp. 627-641 ◽

Cited By ~ 28

Author(s):

Promod K. Mehta ◽

Ankush Raj ◽

Netra Pal Singh ◽

Gopal K. Khuller

Keyword(s):

Nucleic Acid ◽

Infectious Diseases ◽

Real Time ◽

Magnetic Beads ◽

Fold Increase ◽

Biological Molecules ◽

Nucleic Acid Amplification ◽

Pcr Assay ◽

Immunological Research ◽

Sensitive Method

Immuno-PCR (PCR-amplified immunoassay; I-PCR) is a novel ultrasensitive method combining the versatility of ELISA with the sensitivity of nucleic acid amplification of PCR. The enormous exponential amplification power of PCR in an I-PCR assay leads to at least a 102–104-fold increase in sensitivity compared with an analogous ELISA. I-PCR has been used to detect many biological molecules such as proto-oncogenes, toxins, cytokines, hormones, and biomarkers for autoimmune and Alzheimer’s diseases, as well as microbial antigens and antibodies, and it can be adapted as a novel diagnostic tool for various infectious and non-infectious diseases. Quantitative real-time I-PCR has the potential to become the most analytically sensitive method for the detection of proteins. The sensitivity and specificity of a real-time I-PCR assay can be enhanced further with the use of magnetic beads and nanoparticles. This review is primarily focused on the detection of potential viral, bacterial and parasitic antigens by I-PCR assay, thus enabling their application for immunological research and for early diagnosis of infectious diseases.

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Rapid Identification of Candida dubliniensis Using a Species-Specific Molecular Beacon

Journal of Clinical Microbiology ◽

10.1128/jcm.38.8.2829-2836.2000 ◽

2000 ◽

Vol 38 (8) ◽

pp. 2829-2836 ◽

Cited By ~ 44

Author(s):

Steven Park ◽

May Wong ◽

Salvatore A. E. Marras ◽

Emily W. Cross ◽

Timothy E. Kiehn ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Beacon ◽

Random Amplified Polymorphic Dna ◽

Oral Candidiasis ◽

Molecular Beacons ◽

Candida Dubliniensis ◽

Rrna Genes ◽

Enzyme Analysis ◽

Its2 Region ◽

Species Specific

Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference Candida strains was used to develop molecular beacon probes for rapid, high-fidelity identification of C. dubliniensis as well as C. albicans. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely containedC. albicans and a smaller amount of C. dubliniensis, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes BsmAI and NspBII, which cleave recognition sequences within the ITS2 regions of C. dubliniensis and C. albicans, respectively. Molecular beacons are promising new probes for the rapid detection ofCandida species.

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Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5673-5678.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5673-5678 ◽

Cited By ~ 44

Author(s):

Chuanwu Xi ◽

Michal Balberg ◽

Stephen A. Boppart ◽

Lutgarde Raskin

Keyword(s):

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Molecular Beacon ◽

Signal To Noise Ratio ◽

Microfluidic Devices ◽

Molecular Beacons ◽

Target Cells ◽

Signal To Noise ◽

Whole Cells

ABSTRACT DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.

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