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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Singleplex versus Multiplex Assay

PCR is one of the most important scientific advances in the field of Molecular Biology and has radically reduced the time and number of steps required to create large quantities of DNA for use in multiple applications. Where it has had some of its most profound impact has been in the diagnostics field. Not only has PCR increased the speed and accuracy of diagnostics, but it has also been instrumental in the identification and diagnosis of diseases caused by bacteria, viruses, and fungi, many of which are difficult or impossible to identify by conventional methods.
When designing a PCR assay, primer design, reaction conditions, and enzyme selection all need to be taken into consideration. In addition to designing the assay parameters, it is also important to determine what type of assay needs to be designed. For example, depending on the target sequence that is being detected, the PCR assay can either be a singleplex or multiplex assay. Both types of PCR assays are defined below.
  1. Singleplex PCR is used to detect one target sequence of DNA or RNA (as shown in the upper image). This assay could be used to detect a specific virus or bacteria or determine if an individual has a gene of interest.
  2. 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 sets of primers and probes are included to allow more targets/analytes to be detected in a single run.