GenBit
The method employs two-dimensional open-type matrices each node (microreactor) of which (30 or 48) contains stabilised PCR mixture necessary to determine specific object. The mixtures include primers, probes, hot start taq-polymerase and dNTPs required for PCR. In case of working with RNA-containing objects, the mixtures also contain reverse transcriptase to perform preliminary reverse transcription in the microreactor just before the start of the PCR.
The matrices are made on the basis of aluminum or silicon plates (microchips) and are intended for use with "AriaDNA®" microchip nucleic acid amplifier in real time mode.
Special treatment of microchip surface and immobilization/stabilization technology ensure long shelf life of matrices, which remain stable for up to 6 months at room temperature, and reduce the number of necessary manipulations during carrying sample testing (it is just enough to apply extracted DNA/RNA on the matrix).
Evaporation of the sample is prevented by application of sealing liquid (mineral oil), and the combination of the hydrophilic/hydrophobic coatings along the chip surface and microreactors (wells) interferes with any kind of mutual influence and interaction between the wells.
Scheme of the microchip (plate)
Scheme of the pre-filled matrix
PCR effectiveness and matrix uniformity; comparison with traditional format (tubes) of real-time PCR
Uniformity of PCR performance in the matrix microreactors: (a) Pectobacterium carotovorum subsp. carotovorum, Ct = 14.32 ± 0.15, (b) Phytophthora infestans, Ct = 14.89 ± 0.11, (c) PVS, Ct = 13.28 ± 0.05, and (d) PVYO, Ct = 15.97 ± 0.13. IC, Internal Control Sample. DNA/RNA concentration - 1 μg ml−1 (14 wells per sample).
Micromatrix format (right) on AriaDNA® in comparison with tube format of real-time PCR performed on Roche LightCycler 480 II (8 per sample). Testing of the same samples of potato spindle tuber viroid (PSTVd) and potato virus S (PVS) with the use of the same test-systems.
Reproducibility of PCR on pre-filled matrices is controlled by the starting cycle (Ct) of the internal control sample (IC). Results obtained on matrices of various lots manufactured over three years (more than 2000 tests) show that the standard deviation of the C t for IC did not exceed 5%.
Microchip assembly (Al, 30 microreactors). Used as micro-plate to perform real-time PCR with any test-systems, and for the development of applications used on specialised pre-filled PCR matrices with long shelf life.
