STEC qPCR Screening Kit

cat. no. E0480

Description

  • STEC qPCR Screening Kit is a ready to use system for the detection of Shiga toxin-producing Escherichia coli (STEC) infood and environmental samples after enrichment.
  • The kit contains reagents and primers for the amplification of specific regions of E. coli DNA. STECs are identified based on the presence of the Shiga toxin genes (stx1 or stx2, including the stx2f subtype). The intimin gene (eae) is an additional virulence marker. The detection of the STEC amplicons is based on using the specific dual-labelled probes. The stx1/stx2 genes are detected simultaneously in the FAM channel, while the eae gene in the Cy5 channel.
  • In addition, the kit contains a further heterologous amplification system to monitor the proper DNA amplification and identify possible PCR inhibition simultaneously. The internal control system consists of primers and the HEX-labelled probe present in STEC Master Mix and exogenous DNA fragment. The amplification of the inner control does not interfere with the amplification of the specific STEC DNA and confirms PCR reaction correctness.
  • STEC Master Mix contains onTaq DNA Polymerase, optimized reaction buffer, ROX 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 for 15 minutes.
  • ROX passive reference dye included in the STEC 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.
  • DNA isolation should be performed with dedicated, commercially available kits using silica/magnetic beads technology.