Amplicon biotinylated

Fig. 30.4. Evaluation of 2.9 pmol of IS200 amplicon using a genosensor based on dry-adsorption of the target on GEC transducer 68.2 pmol of biotinylated probe 9.00 /ig of enzyme conjugate. The nonspecific adsorption signal is shown in black (more details in Ref. [2]) (N — 3).

When testing DNA amphfied samples, the amplicons were denatured to dissociate the dsDNA into ssDNA, using the approaches reported in Sect. 2.3 of this chapter. In Fig. 5 the response obtained with the different denaturation procedures using the biotinylated probe and streptavidin-modified surfaces is shown. A good reproducibility was found with PCR-amplified material (CV% 6 ( = 3)). 

PCR-ELISA is a hybrid assay combining PCR as a first step and using ELISA as a detection system for the amplicons (Figure 9.1). The PCR step produces biotinylated amplicons that are bound further to a streptavidin-coated microtiter plate. The amplicons are denatured to produce single-stranded PCR products, and only the biotinylated strand stays in the well of the microtiter plate. A FITC-Iabeled probe is bound to the PCR strand, a step that adds an extra level of specificity to the assay. An enzyme-labeled anti-FITC antibody is used to produce a colorimetric signal that can be measured by an ELISA reader. This method has been applied successfully to the detection of <10 ppm hazelnut in processed foods and food ingredients, which eliminated the potential for cross-reactivity observed in ELISA (Holzhauser et al., 2002). 

The approach using PNA hybridisation probes for MALDI-TOF mass spectrometric analysis is comprised of the following steps (Figure lb) immobilisation of biotinylated target DNA (e.g. a PCR amplicon) by binding to streptavidin coated magnetic beads dissociation and removal of the non-biotinylated... 

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