Internal positive/amplification control

AU real-time PCR systems in Tables 5.4 and 5.5 are compatible, operate using the same tanperature protocol (95°C lOmin 40x95 C 10s, tiO C 55s 20°C >), and detect the products in the RAM channel, which is available in most real-time PCR cyclers. The composition of the PCR mix as well as the DNA sequences and the application of the internal positive/amplification control (IPC or lAC) in the HEX channel are described by Brandi and Hutzler (Brandi, 2006 Hutzler, 2009). Qualitative results for certain strains of the Saccharomyces sensu stricto complex analyzed with the realtime PCR systems in Tables 5.4 and 5.5 are shown in Table 5.6. 

Castelain, S., V. Descamps, V. Thibault, C. Francois, et al. TaqMan Amplification System with an Internal Positive Control for HCV RNA Quantitation. Journal of Clinical Virology 31 no. 3 (2004) 227-234. 

Internal positive controls (IPCs) can also be used to identify the presence of PCR inhibitors. IPCs are definitively useful for detecting false-negative results however, they cannot be used to determine a precise measure of inhibition strength when template samples are marginally compromised. Comparing the amplification efficiencies of clean standards with those of unknown samples is a statistically sound method that can be used in conjunction with IPCs when amplifications are successful but are compromised, producing erroneous quantification results. 

In the original ASA protocols, the mutant and normal PCR primers were separated into two reactions, so that lack of amplification could occur in one PCR reaction depending on the sequence present in a test sample. This is not ideal for a diagnostic test due to the possible misinterpretation of a false negative result, and the ASA protocol is usually modified to be a multiplex reaction that includes a positive internal control in each PCR reaction. For example, an ASA assay has been developed, which detects 12 common CFTR mutations simultaneously (17). However in this assay, two reactions must still be run in parallel for every sample to be analyzed, since the mutant and normal products produced are the same size and so must be physically separated in order to be distinguished. 

Direct analysis of PCR products by electrophoresis (without enzyme digestion) can also allow the final interpretation of an assay in which the presence of an amplification product is directly diagnostic (e.g., for the presence of a bacterium, virus, or fungus, in a specimen). The specificity of the amplification reaction is verified by the known size of the fragment. Internal negative and positive controls are employed to control for potential contamination and to establish detection sensitivity. 

A key consideration with molecular assays is controlling for inhibition of amplification. This is particularly critical for the detection of MTb nucleic acid from respiratory specimens, because these specimen types often contain blood or glycoprotein, which can inhibit amplification. The Amplicor assay contains an internal control, and a negative result cannot be reported unless the internal control is detected. The MTD test does not contain an inhibition control, though many laboratories include such a control by adding a positive control material to a second aliquot of the clinical specimen. 

Primers for the Amplification of Some Housekeeping Genes as Positive Internal Control... 

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