Quantification of PCR products using microparticles and flow cytometry

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.