Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.
Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.|Hahn-Schickard-Institut für Mikroanalysesysteme, Freiburg, Germany.
Simon
S
Wadle
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.
Michael
M
Lehnert
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.|Hahn-Schickard-Institut für Mikroanalysesysteme, Freiburg, Germany.
Friedrich
F
Schuler
Food Control Authority of the Canton of Zürich, Zürich, Switzerland.
René
R
Köppel
Department of Medical Microbiology and Hygiene, University Hospital Freiburg, Freiburg, Germany.
Annerose
A
Serr
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.|Hahn-Schickard-Institut für Mikroanalysesysteme, Freiburg, Germany.|BIOSS-Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
Roland
R
Zengerle
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.|Hahn-Schickard-Institut für Mikroanalysesysteme, Freiburg, Germany.
Felix
F
von Stetten
D000818:Animals; D004247:DNA / Q000032:analysis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D005456:Fluorescent Dyes / Q000737:chemistry*; D005502:Food / Q000145:classification; D005504:Food Analysis / Q000379:methods*; D057230:Limit of Detection; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D004247:DNA
MRSA; food panel; mediator probe PCR; multiplex PCR; universal reporter