| 摘要 |
The invention relates to molecular biology, medicine and biotechnology, in particular to a polymerase chain reaction method and a device for the implementation thereof with the real-time recording of reaction product accumulation. The problem addressed by the invention is solved using a method and a corresponding device for determining nucleic acids by real-time polymerase chain reaction, which method involves introducing liquid nucleic acid-containing samples into reaction areas on the upper surface of a thermally conductive microchip substrate, insulating said samples from the atmosphere, reacting the nucleic acid in a sample with polymerase chain reaction components while thermocycling the samples and removing heat via the outer surface of the microchip, carrying out the fluorescent determination of a change in quantity of the polymerase chain reaction products during the thermocycling process and determining the quantity of initial nucleic acid in the samples according to the dynamics of the fluorescence signal growth. The method is characterized in that a microchip is used which has a thermally conductive substrate made of a thermally conductive material with a thermal conductivity factor greater than 1 W/cm K and a thermal diffusion coefficient greater than 0.6 cm/s, wherein the reaction areas on the microchip surface are separated from the thermally conductive substrate by a layer of passivation material which is covalently bound to the surface of the thermally conductive material and the introduced samples are insulated from the atmosphere by a layer of water-immiscible liquid which is retained on the upper surface of the thermally conductive substrate with the aid of a peripheral barrier; furthermore, the relationship between the total thermal mass of the microchip, together with the introduced samples and the layer of water-immiscible liquid, and the thermal conductivity of the microchip substrate is equal to or less than 0.04 s. The technical result consists in reducing analysis time and increasing the reliability, fidelity, efficiency and cost-effectiveness of analysis. |
