Multiplex analytical system for DNA determination by real-time PCR

A microchip analytical system has been developed to implement real-time polymerase chain reaction (RT-PCR) with simultaneous detection of two fluorescence dyes. This system allows performing a multiplex PCR in one microreactor. The microchip is made of heat-conducting plate covered with water-immiscible fluid and contains 16 or 30 microreactors, so that samples of 1-2 µl volume can be loaded manually. Surface modification procedure has been developed for aluminum and silicon microchip to define hydrophilic and hydrophobic areas in order to lock sample inside the microreactor during thermocycling and to prevent PCR inhibition and cross-contamination. Practical applications of the developed analytical system using commercial multiplex PCR reagent kits are demonstrated. Several advantages for genetically modified organisms screening are achieved such as short analysis time (20 min), low reagent consumption (1-2 µl per reaction), low number of operations and high reliability of results obtained with internal control reagents, all these advantages define a wide practical applicability of the microchip RT-PCR system.