Multiplex quantitative competitive polymerase chain reaction based on a multianalyte hybridization assay performed on spectrally encoded microspheres

Quantitative polymerase chain reaction (PCR) may be performed by two general approaches, namely, real-time PCR and quantitative competitive PCR (QC-PCR). QC-PCR makes use of the concept of a DNA competitor, which is the gold standard approach to circumvent the problem of the variation of amplification efficiency. However, QC-PCR in its classical form is a low-throughput method since it requires titration of each sample with the competitor followed by electrophoresis. The throughput of QC-PCR has been improved by capillary electrophoresis and microtiter well-based hybridization assays. The present work introduces a multiplex QC-PCR method, which is based on a multianalyte hybridization assay that is performed on spectrally encoded microspheres. The DNA competitors use the same primers and have equal size with their corresponding target DNA sequences but differ in a short (24 bp) centrally located sequence. Following multiplex PCR, biotinylated amplification products from all DNA targets and competitors are heat-denatured and hybridized with oligonucleotide probes, which are attached to addressable sets of fluorescent microspheres. The hybrids react with a streptavidin-phycoerythrin conjugate. The microspheres are then analyzed by flow cytometry employing two lasers. A red laser line is used for classification of the microspheres, and a green line excites phycoerythrin, whose fluorescence is related to the concentration of the analyte DNA. As a model, we have developed a multiplex quantitative competitive PCR assay for four targets. The amplification products from targets and competitors (a total of 8 DNA fragments) are determined simultaneously by the multianalyte hybridization assay. The limits of quantification for the hybridization assay of all amplified DNA fragments are below 13 pM. The multiplex quantitative competitive PCR assay detects similar to 500 copies from each target DNA. To our knowledge, the proposed method is the only approach to quantitative PCR that offers such a high potential for multiplexing.