TY - JOUR
T1 - A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.
AU - Horejsh, Douglas
AU - Martini, Federico
AU - Poccia, Fabrizio
AU - Ippolito, Giuseppe
AU - Di Caro, Antonino
AU - Capobianchi, Maria R.
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=19744383959&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=19744383959&partnerID=8YFLogxK
M3 - Article
C2 - 15659574
AN - SCOPUS:19744383959
VL - 33
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 2
ER -