Multiplex polymerase chain reaction

Multiplex PCR is a form of application of the diagnostic PCR. Initially the PCR especially for the amplification ( multiplication ) of DNA was used for sequencing (determination of base sequence ) of the amplified DNA. With the progress of the sequencing projects, it became possible to use the PCR as a diagnostic method. With knowledge of the base sequence in the genome of an organism, it was possible to identify the specific sequence for this organism or disease related sequence changes. A diagnostic PCR is then developed on these specific sequences. Such a PCR for the detection of a portion of the genome is also called a singleplex PCR or single -PCR. For many diseases, but can be the cause of different viruses or bacteria or genomic changes. Therefore, it is to determine the cause of the condition necessary to carry out multiple singleplex PCR detection. Under these conditions, a multiplex PCR is useful.

Principle

In a multiplex PCR, the single PCR method are not realized in separate PCR reactions, but all together in a PCR reaction. So that the burden is to determine the cause of the disease, particularly the cost and time is reduced. A multiplex PCR is thus a PCR approach with the potential for the detection of more than one section of the genome. The development of a multiplex PCR is associated with an increased cost. Due to the increased number of primers and probes ( when configured as a real-time PCR ) interactions between these oligonucleotides are much more likely and occur much more frequently in the real world on. These interactions would reduce the sensitivity of the multiplex PCR and must therefore be excluded.

Stand

Under real conditions is very difficult to completely exclude these interactions between the oligonucleotides, which is why sensitive multiplex PCR applications are currently mainly duplex PCR. And this in turn is combined very often proof to be determined genome sequence with an internal amplification control. An amplification is required for many diagnostic PCR applications in order to avoid false negative results. False-negative results would occur if the DNA to be investigated inhibitors were included for the PCR detection. In reality, these are especially inhibitors of the enzymatic activity of Taq - polymerase, such as iron ions from the red blood cells. The amplification is a PCR detection, which detects the absence of inhibitors of the PCR. For a second PCR reaction is constructed and added to the PCR mixture with a small amount of template DNA for this PCR. With a negative result for the detection of the investigated genome sequence, this amplification must be positive in order to confirm the absence of inhibitors, and thus the accuracy of the negative result. Although duplex PCR reactions were most widespread, as are methods with up to four certificates per PCR approach no longer a rarity.

Reference

Under marketing point some companies sell as a multiplex PCR detection kits that contain several separate PCR reactions. This is not a real multiplex PCR.

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Because of the large number of real clinical problems with a variety of possible causes is to start from a wider dissemination of multiplex PCR method. Exemplary are the key areas such as sepsis analysis, the diagnosis of respiratory diseases, cancer diagnosis, the detection of genetically modified organisms ( GMOs) or the cause of diarrhea. It is to be expected especially with a further increase in the multiplex number and detection by real-time PCR.