Primer-probe systems

TABLE 7.1 Primer-Probe Systems Used and Amplicon Length Expected"... 

FIGURE 7.6 Fluorescence curves for samples of cattle, pig, lamb, goat, chicken, turkey, and duck using the MYw primer-probe system. C/s are listed in Table 7.6. 

The assay consists of four t3 pes of controls positive, cutoff, extraction control (water treated like sample), and the IPC system. The positive control made either from meat DNA or from plasmid DNA containing the respective target sequence aims to be an amplification control to demonstrate the functionality of the primer-probe system. The cut off control contains alow copy number of target DNA, close to the LOD of the primer-probe systems. Based on different levels of controls, the entire system guarantees a high grade of reliability and robustness. The use of plasmid DNA is an innovative tool that avoids problems related to DNA isolated from species of different origins. Furthermore, plasmid DNA can be reproduced easily with the same quality, as often as necessary. 

Tai t-specificity Primer Probe System name Primer sequence (S - 3 ) Reference... 

Mixed Mechanism Probes. Several probe systems appear to function by both hydrolysis and hybridization mechanisms. These include hairpin probes, self-probing amplicon primers, and displacement probes. A hairpin probe functions similarly to a hairpin primer in that it is designed to increase in fluorescence when the distance between the quencher and the reporter increases upon target hybridization (see Figure 37-24, row five). Similarly, primers that... 

Real-time PCR also requires a more complex thermocycler than does traditional PCR because a light source and a fluorescence detection system are required. Adding to the expense of real-time PCR are the costs of SYBR green or labeled primer/probe sets, a computer, and an analytical software package. 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

Many different fluorescent reporter systems are used in realtime PCR, and some of the more common ones are shown in Figure 37-24. Many methods use probes with sequences complementary to the target. Others rely on the specificity afforded by PCR primers, and some have the additional option of melting analysis to verify the melting temperature of the probe or product. 

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