PCR is one of the most important scientific advances in molecular biology and has radically reduced the time and number of steps required to create large quantities of DNA for use in multiple applications. It has had some of its most profound impacts in the diagnostics field. Not only has PCR increased the speed and accuracy of diagnostics, but it has also been instrumental in identifying and diagnosing diseases caused by bacteria, viruses, and fungi, many of which are difficult or impossible to identify by conventional methods.
Primer design, reaction conditions, and enzyme selection must be considered when designing a PCR assay. In addition to creating the assay parameters, it is also essential to determine what type of assay needs to be developed. For example, depending on the target sequence being detected, the PCR assay can either be a singleplex or multiplex assay. Both types of PCR assays are defined below.
- Singleplex PCR detects one DNA or RNA target sequence (as shown in the upper image). This assay could detect a specific virus or bacteria or determine whether an individual has a gene of interest.
- Multiplex PCR is used to detect two or more target sequences of DNA or RNA simultaneously (shown in the lower image) using a single sample preparation and amplification. Multiple primers and probes are included to allow more targets/analytes to be detected in a single run.
