A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.
Identifieur interne : 004C77 ( Main/Merge ); précédent : 004C76; suivant : 004C78A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.
Auteurs : Douglas Horejsh [Italie] ; Federico Martini ; Fabrizio Poccia ; Giuseppe Ippolito ; Antonino Di Caro ; Maria R. CapobianchiSource :
- Nucleic acids research [ 1362-4962 ] ; 2005.
Descripteurs français
- KwdFr :
- ADN viral (analyse), Acides nucléiques (analyse), Colorants fluorescents (), Cytométrie en flux, Hybridation d'acides nucléiques (), Microsphères, Mésappariement de bases, Oligonucléotides (analyse), Sondes d'acide nucléique (), Streptavidine (), Techniques de diagnostic moléculaire, Virus du SRAS (génétique).
- MESH :
English descriptors
- KwdEn :
- Base Pair Mismatch, DNA, Viral (analysis), Flow Cytometry, Fluorescent Dyes (chemistry), Microspheres, Molecular Diagnostic Techniques, Nucleic Acid Hybridization (methods), Nucleic Acid Probes (chemistry), Nucleic Acids (analysis), Oligonucleotides (analysis), SARS Virus (genetics), Streptavidin (chemistry).
- MESH :
- chemical , analysis : DNA, Viral, Nucleic Acids, Oligonucleotides.
- chemical , chemistry : Fluorescent Dyes, Nucleic Acid Probes, Streptavidin.
- genetics : SARS Virus.
- methods : Nucleic Acid Hybridization.
- Base Pair Mismatch, Flow Cytometry, Microspheres, Molecular Diagnostic Techniques.
Abstract
Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.
DOI: 10.1093/nar/gni015
PubMed: 15659574
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pubmed:15659574Le document en format XML
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<author><name sortKey="Martini, Federico" sort="Martini, Federico" uniqKey="Martini F" first="Federico" last="Martini">Federico Martini</name>
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<author><name sortKey="Poccia, Fabrizio" sort="Poccia, Fabrizio" uniqKey="Poccia F" first="Fabrizio" last="Poccia">Fabrizio Poccia</name>
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<author><name sortKey="Ippolito, Giuseppe" sort="Ippolito, Giuseppe" uniqKey="Ippolito G" first="Giuseppe" last="Ippolito">Giuseppe Ippolito</name>
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<series><title level="j">Nucleic acids research</title>
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<term>Fluorescent Dyes (chemistry)</term>
<term>Microspheres</term>
<term>Molecular Diagnostic Techniques</term>
<term>Nucleic Acid Hybridization (methods)</term>
<term>Nucleic Acid Probes (chemistry)</term>
<term>Nucleic Acids (analysis)</term>
<term>Oligonucleotides (analysis)</term>
<term>SARS Virus (genetics)</term>
<term>Streptavidin (chemistry)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>ADN viral (analyse)</term>
<term>Acides nucléiques (analyse)</term>
<term>Colorants fluorescents ()</term>
<term>Cytométrie en flux</term>
<term>Hybridation d'acides nucléiques ()</term>
<term>Microsphères</term>
<term>Mésappariement de bases</term>
<term>Oligonucléotides (analyse)</term>
<term>Sondes d'acide nucléique ()</term>
<term>Streptavidine ()</term>
<term>Techniques de diagnostic moléculaire</term>
<term>Virus du SRAS (génétique)</term>
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<term>Oligonucleotides</term>
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<term>Nucleic Acid Probes</term>
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<term>Oligonucléotides</term>
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<term>Microspheres</term>
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<term>Cytométrie en flux</term>
<term>Hybridation d'acides nucléiques</term>
<term>Microsphères</term>
<term>Mésappariement de bases</term>
<term>Sondes d'acide nucléique</term>
<term>Streptavidine</term>
<term>Techniques de diagnostic moléculaire</term>
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<front><div type="abstract" xml:lang="en">Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.</div>
</front>
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