External and internal controls

All real-time PCR assays should include positive and negative controls and reliability of an assay is intimately associated with the quality of the assay controls. Some substances found in clinical samples can inhibit PCR, possibly via effecting binding and/or polymerization activity of DNA poly-merases, such as bile salts and complex polysaccharides of feces and heme of blood (Nolte, 2004; Monteiro et al., 1997; Al-Soud and Radstrome, 2001). Degradation of target nucleic acid, sample processing errors, thermal cycler malfunction, and carry-over contamination are some of the other reasons that may cause false-negative results (Nolte, 2004).

External controls are simple approaches to detect inhibitors but addition of an external control to a separate reaction increases the costs and does not always work for large batch sizes (Nolte, 2004). Usually, an external control is created by using a cloned amplicon, a portion of the target organisms' genome, or simply the purified amplicon itself (Mackay, 2004). The data generated from individual amplification of the known dilution series of external control create the basis of the standard curve which is used for analyzing the unknown sample content. Internal controls, which are spiked into clinical patient sample, provide an accurate way to check the integrity of all amplification steps. They should imitate the target sequence and if they are not amplified, that should indicate the inhibition of the PCR analysis of clinical patient sample (Nolte, 2004; Cockerill, 2003). Melting curve analyses can be another important quality control feature that confirms PCR amplicons as the correct amplification product and can discriminate base pair differences in target DNA, such as mutations (Cockerill, 2003; Wittwer and Kusukawa, 2004).

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