Multiplex-microsphere-quantitative polymerase chain reaction: Nucleic acid amplification and detection on microspheres

Fang Liang, Richard Lai, Neetika Arora, Kang Liang Zhang, Che-Cheng Yeh, Graeme R. Barnett, Paul Voigt, Simon R. Corrie, Ross T. Barnard

Research output: Contribution to journalArticleResearchpeer-review

7 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)23 - 30
Number of pages8
JournalAnalytical Biochemistry
Volume432
Issue number1
DOIs
Publication statusPublished - 1 Jan 2013
Externally publishedYes

Keywords

  • Polymerase chain reaction
  • Real time
  • Microsphere
  • Quantitative
  • Multiplex
  • Luminex

Cite this