There are a multitude of different uses for which multiplex real-time PCR can be employed. In a clinical setting, this technology has been most prominently used in the infectious disease field, in which case it is highly beneficial to be able to simultaneously identify and detect multiple pathogens from a single specimen. This approach has become commonplace in many laboratories, with assays that can be designed to be qualitative or quantitative. Quantitative multiplex reactions may involve amplification of multiple targets that are quantified using external calibration curves. Alternately, relative quantification can be calculated using products that are coamplified in the same reaction. This method typically compares or normalizes the target of interest to a calibrated standard or to a housekeeping gene. This relative quantitation is also an example of another separate use for multiplexing. With respect to relative quantitation, greater accuracy is achieved using multiplexing because identical reaction conditions are present for both targets being compared (they are in the same reaction tube).
The addition of an internal positive control (IPC) to a reaction is another common use for multiplexing in a clinical setting. The inclusion of an IPC monitors the efficiency of each individual PCR reaction. The IPC can be included at the point of specimen collection to monitor sample stability, it can be added just prior to nucleic acid extraction to monitor extraction efficiency, or it can be added solely to the PCR mastermix to determine whether inhibitory substances are present following extraction.
In addition to infectious disease applications, multiplex real-time PCR is also extensively used in the fields of molecular genetics and oncology. For instance, multiplex techniques are used to detect multiple SNPs in a single reaction and to do allelic discrimination experiments. In addition, relative quantitation is used for gene dosing determinations, and it is also used to monitor gene expression; reverse-transcriptase PCR can be used on mRNA to determine the expression levels of tumor markers or to determine the response of specific genes to drug therapy. Some examples of real-time PCR applications are listed in Table 1. This is but a small sampling of the many uses for multiplex PCR, but a more complete listing and description is beyond the scope of this work.
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