Multiplex on-Chip PCR with Direct Detection of Immobilized Primer Elongation

A variant of multiplex PCR on a chip with direct detection of immobilized primer elongation has been developed. Detection is performed by determining the fluorescence signal of labeled dUTPs incorporated in the growing chain. This approach creates an elongated immobilized primer with a covalently bound fluorescent label. The product of a primer elongation carries several fluorescent labels (depending on the AT composition and length of the PCR product), which allows the complete removal of all the components of the mixture after PCR and thus dramatically reduces the background signal and increases the sensitivity of analysis on hydrogel microchips. The method was successfully applied for differential detection of important bacterial pathogens of human pneumonia Staphylococcus aureus and Streptococcus pneumoniae, including testing of the one analyzed sample for the both pathogens.