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Internal amplification controls (IAC) have been developed to detectPCRinhibitors (Casas et al. 1997) and have become an important quality control measure for diagnostic PCR assays (Malorny et al. 2003b). Sample or processed templates are spiked with the control DNA (Malorny et al. 2003a). This control DNA or IAC is generally derived through an internal engineered deletion within the cloned amplicon (Abdulmawjood et al. 2002) (Figure 3). Spiked with the IAC, the PCR primers would produce an amplicon with a size expected for the engineered deletion. A sample containing the targeted pathogen will produce two amplicons, one corresponding to the size expected for the pathogen and the other smaller amplicon expected for the IAC (Abdulmawjood et al. 2002). Inclusion of an IAC into the diagnostic PCR test has become an important part in harmonization of PCR-based detection protocols for foodborne pathogens (Malorny et al. 2003a). With the advance of real-time PCR, IAC have been adapted to these newer diagnostic tests as part of a TAQMAN PCR (Rodr´ıguez-L´azaro et al. 2005). In this case, the internal oligonucleotide probe is directed to unique sequences obtained by the deletion of internal sequences present in the IAC.With inclusion of different colored fluorescent dyes in the labeling of pathogen-specific oligonucleotide probes and IAC probes, real-time thermocyclers can differentiate between the two signals obtained with either probe (Rodr´ıguez-L´azaro et al. 2005). |
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