A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.
Identifieur interne : 002929 ( PubMed/Curation ); précédent : 002928; suivant : 002930A 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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Capobianchi</name></author></analytic><series><title level="j">Nucleic acids research</title><idno type="eISSN">1362-4962</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Pair Mismatch</term><term>DNA, Viral (analysis)</term><term>Flow Cytometry</term><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></keywords><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></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>DNA, Viral</term><term>Nucleic Acids</term><term>Oligonucleotides</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Fluorescent Dyes</term><term>Nucleic Acid Probes</term><term>Streptavidin</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ADN viral</term><term>Acides nucléiques</term><term>Oligonucléotides</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Nucleic Acid Hybridization</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Pair Mismatch</term><term>Flow Cytometry</term><term>Microspheres</term><term>Molecular Diagnostic Techniques</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Colorants fluorescents</term><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></keywords></textClass></profileDesc></teiHeader><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></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15659574</PMID><DateCompleted><Year>2005</Year><Month>02</Month><Day>16</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1362-4962</ISSN><JournalIssue CitedMedium="Internet"><Volume>33</Volume><Issue>2</Issue><PubDate><Year>2005</Year><Month>Jan</Month><Day>19</Day></PubDate></JournalIssue><Title>Nucleic acids research</Title><ISOAbbreviation>Nucleic Acids Res.</ISOAbbreviation></Journal><ArticleTitle>A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.</ArticleTitle><Pagination><MedlinePgn>e13</MedlinePgn></Pagination><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Horejsh</LastName><ForeName>Douglas</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Rome, Italy. horejsh@inmi.it</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Martini</LastName><ForeName>Federico</ForeName><Initials>F</Initials></Author><Author ValidYN="Y"><LastName>Poccia</LastName><ForeName>Fabrizio</ForeName><Initials>F</Initials></Author><Author ValidYN="Y"><LastName>Ippolito</LastName><ForeName>Giuseppe</ForeName><Initials>G</Initials></Author><Author ValidYN="Y"><LastName>Di Caro</LastName><ForeName>Antonino</ForeName><Initials>A</Initials></Author><Author ValidYN="Y"><LastName>Capobianchi</LastName><ForeName>Maria R</ForeName><Initials>MR</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D023362">Evaluation Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2005</Year><Month>01</Month><Day>19</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Nucleic Acids Res</MedlineTA><NlmUniqueID>0411011</NlmUniqueID><ISSNLinking>0305-1048</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004279">DNA, Viral</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005456">Fluorescent Dyes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D015341">Nucleic Acid Probes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009696">Nucleic Acids</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009841">Oligonucleotides</NameOfSubstance></Chemical><Chemical><RegistryNumber>9013-20-1</RegistryNumber><NameOfSubstance UI="D019809">Streptavidin</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D020137" MajorTopicYN="N">Base Pair Mismatch</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004279" MajorTopicYN="N">DNA, Viral</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005434" MajorTopicYN="Y">Flow Cytometry</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005456" MajorTopicYN="N">Fluorescent Dyes</DescriptorName><QualifierName UI="Q000737" 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