Description

• Duplex TBEV qRT-PCR Kit is a ready-to-use system for thedetection of TBEV RNA in animal tissue samples (tick, human).
• The kit detects all three TBEV subtypes: Western European, Siberian and Far Eastern subtype.
• Duplex TBEV qRT-PCR Kit contains reagents and primers for the amplification ofa region of the TBEV genome. The detection of the TBEV amplicon is based on using the specific FAM-labelled probe.
• In addition, the kit contains a second heterologous amplification system to monitor the RNA isolation procedure and identify possible PCR inhibition simultaneously. The internal control system consists of: primers, the HEX-labelled probe present in TBEV Master Mix and the exogenous nucleic acid fragment (IC-internal control). The amplification of the inner control does not interfere with the amplification of the specific TBEV RNA and confirms RNA isolation and/or PCR reaction correctness.
• IC-internal control can be added at the RNA isolation stage (point a) – then it is both a control of the RNA isolation and PCR inhibition, or only at the PCR stage (point b) – to assess the quality of the purified RNA and possible PCR inhibition.

1. Add IC to the lysis buffer or to the mixture of lysis buffer and sample material at a ratio of 0.1 μl per 1 μl elution volume. For example, 5 μl of IC should be added initially using 50 μl elution volume. It is recommended to use carrier RNA when isolating viral RNA. Carrier RNA increases the efficiency of RNA binding to silica membranes in the presence of small amounts of genetic material in the sample and reduces viral RNA degradation.
2. Add 0.5 µl of IC for every 20 µl of PCR reaction.

• TBEV Master Mix contains smART reverse transcriptase, onTaq DNA Polymerase, optimized reaction buffer, dNTPs, and the ROX dye.
• onTaq DNA Polymerase is a modified “hot start” enzyme that is blocked at moderate temperatures and allows room temperature reaction setup.
• ROX passive reference dye included in the TBEV Master Mix allows fluorescence normalization on certain cyclers. The use of ROX passive reference dye is necessary for all real-time PCR cyclers from Applied Biosystems and optional for cyclers from Agilent. ROX compensates for variations of fluorescent signal between wells due to slight differences in reaction volume and fluorescence fluctuations. ROX is not involved in PCR reaction and does not interfere with real-time PCR on any instrument.
• RNA isolation from animal tissues should be performed with dedicated, commercially available kits using silica/magnetic beads technology.