- ASFV qPCR Detection Kit is a ready-to-use system for the detection of DNA from African Swine Fever Virus in serum, plasma, blood, tissue and swab samples from pig and wild boar.
- The kit contains reagents and primers for the amplification of a highly conserved ASFV p72 gene fragment. The detection of the ASFV amplicon is based on using the specific FAM-labelled probe.
- In addition, the kit contains a second heterologous amplification system to monitor DNA isolation and PCR reaction correctness. The inner control consists of primers and the HEX-labelled probe for porcine ACTB gene coding β-actin and present within the sample.
- ASFV qPCR Master Mix (2x) contains onTaq DNA Polymerase, optimized reaction buffer, dNTPs all necessary primers and probes required and ROX passive reference dye.
- 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 at 95°C for 15 minutes.
- ROX passive reference dye included in the master mix allows fluorescence normalization on certain cyclers. The use of ROX 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.
- The positive control includes the targeted viral DNA and porcine ACTB gene region and serves to prove functionality of the ASFV assay. CT for the ASFV target in the FAM channel should be: 30±3, while for the ACTB target in the HEX channel should be: 29±3.
- Prior to real-time PCR, viral DNA must be extracted from the starting material with dedicated, commercially available kits using silica /magnetic beads technology.