Cargando…

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 pr...

Descripción completa

Detalles Bibliográficos
Autores principales: Sukla, Soumi, Mondal, Prasenjit, Biswas, Subhajit, Ghosh, Surajit
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699591/
https://www.ncbi.nlm.nih.gov/pubmed/34940236
http://dx.doi.org/10.3390/bios11120479
_version_ 1784620550036914176
author Sukla, Soumi
Mondal, Prasenjit
Biswas, Subhajit
Ghosh, Surajit
author_facet Sukla, Soumi
Mondal, Prasenjit
Biswas, Subhajit
Ghosh, Surajit
author_sort Sukla, Soumi
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8699591
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-86995912021-12-24 A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons Sukla, Soumi Mondal, Prasenjit Biswas, Subhajit Ghosh, Surajit Biosensors (Basel) Article 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. MDPI 2021-11-26 /pmc/articles/PMC8699591/ /pubmed/34940236 http://dx.doi.org/10.3390/bios11120479 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sukla, Soumi
Mondal, Prasenjit
Biswas, Subhajit
Ghosh, Surajit
A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title_full A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title_fullStr A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title_full_unstemmed A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title_short A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons
title_sort rapid and easy-to-perform method of nucleic-acid based dengue virus diagnosis using fluorescence-based molecular beacons
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699591/
https://www.ncbi.nlm.nih.gov/pubmed/34940236
http://dx.doi.org/10.3390/bios11120479
work_keys_str_mv AT suklasoumi arapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT mondalprasenjit arapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT biswassubhajit arapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT ghoshsurajit arapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT suklasoumi rapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT mondalprasenjit rapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT biswassubhajit rapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons
AT ghoshsurajit rapidandeasytoperformmethodofnucleicacidbaseddenguevirusdiagnosisusingfluorescencebasedmolecularbeacons