- Duplex Babesia qPCR Kit is a ready-to-use system for thedetection of protozoan parasite Babesia spp. in animal tissue samples (tick, mammal).
- The kit contains reagents and primers for the amplification ofa region of the Babesia genome. The detection of the Babesia amplicon is based on using the specific FAM-labelled probe.
- In addition, the kit contains a second heterologous amplification system to monitor the DNA isolation procedure and identify possible PCR inhibition simultaneously. The internal control system consists of: primers, the HEX-labelled probe present in Duplex Babesia Master Mix and exogenous DNA fragment (IC-internal control). The amplification of the inner control does not interfere with the amplification of the specific Babesia DNA and confirms DNA isolation and/or PCR reaction correctness.
- IC-internal control can be added at the DNA isolation stage (point a) – then it is both a control of the DNA isolation and PCR inhibition, or only at the PCR stage (point b) – to assess the quality of the purified DNA and possible PCR inhibition.
- Add IC to the lysis buffer or to the mixture of lysis buffer and sample material (do not add to the sample material directly) at a ratio 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 DNA from cell-free body fluids and material low in DNA/RNA content. Carrier RNA increases the efficiency of DNA binding to silica membranes in the presence of small amounts of genetic material in the sample.
- Add 0.5 µL of IC for every 20 µL of PCR reaction.
- Duplex Babesia Master Mix contains onTaq DNA Polymerase, optimized reaction buffer and dNTPs.
- onTaq DNA Polymerase is a modified “hot start” enzyme that is blocked at moderate temperatures and allows room temperature reaction setup.
- The polymerase activity is restored during the initial denaturation step when amplification reactions are heated at95°C for 15 minutes.
- ROX passive reference dye included in the Duplex Babesia 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 Stratagene. 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.
- DNA isolation from animal tissues should be performed with dedicated, commercially available kits using silica/magnetic beads technology.
List of Babesia species detected with Duplex Babesia qPCR Kit
B. annae, B. bigemina, B. bovis, B. caballi, B. canis, B. capreoli, B. conradae, B. crassa, B. divergens, B. duncani, B. equi, B. felis, B. gibsoni, B. hongkongensis, B. lengau, B. leo, B. microti, B. motasi, B. occultans, B. odocoilei, B. orientalis, B.ovata, B. ovis, B. rodhaini, B. vulpes