title: Multiplex-microsphere-quantitative polymerase chain reaction: nucleic acid amplification and detection on microspheres.
abstract: We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.
authors.affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, University of Queensland, St. Lucia, Queensland 4072, Australia.
authors.forename: Fang
authors.initials: F
authors.lastname: Liang
authors.forename: Richard
authors.initials: R
authors.lastname: Lai
authors.forename: Neetika
authors.initials: N
authors.lastname: Arora
authors.forename: Kang Liang
authors.initials: KL
authors.lastname: Zhang
authors.forename: Che-Cheng
authors.initials: CC
authors.lastname: Yeh
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Barnett
authors.forename: Paul
authors.initials: P
authors.lastname: Voigt
authors.forename: Simon R
authors.initials: SR
authors.lastname: Corrie
authors.forename: Ross T
authors.initials: RT
authors.lastname: Barnard
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D057230:Limit of Detection; D008863:Microspheres; D060885:Multiplex Polymerase Chain Reaction; D021141:Nucleic Acid Amplification Techniques
chemical_list: D005456:Fluorescent Dyes