The Disadvantages of Multiplex PCR can be Rectified by Using a Second Nested PCR Step

Advantages of Multiplex PCR:

  • Multiplex qPCR allows more information from a single reaction, particularly important when input sample is limited;
  • Reduces reaction costs, multiplex reaction costs are closer to a one singleplex reaction than four independent singleplex reactions.


Disadvantages of Multiplex PCR:

  • Multiplex reactions increase the chances of false positive signals;
  • Biased amplification: some amplicons are nearly always favored over others. It is often a significant effort to develop a 1-step multiplex PCR panel where all targets amplify with similar efficiency;
  • When doing single step multiplex qPCR, separate fluorescent labels are needed for each amplicon (and only up to 4 typically).



Achieve the advantages and avoid the disadvantages of Multiplex Nested PCR by using the Alluvia System

Advantages and Disadvantages of Multiplex Nested PCR

Advantages of Nested PCR:

  • If nonspecific products are amplified because of mispriming by the first set of primers (outer primers), it is very unlikely that the same nonspecific region would be recognized and sequentially amplified by the second primer set, so specificity is promoted by the second set of primers;
  • Increases yield of the desired target from a limited amount of input DNA;
  • Reducing PCR complexity in the second nested round of PCR (by running multiple reactions) reduces both amplicon bias and the need for separate labels.


Disadvantages of Nested PCR:

  • The products of the first PCR step are diluted and used in the second PCR step. This creates a serious PCR product contamination risk;
  • Not widely deployed in clinical settings because, in most systems, it is an open-tube procedure that is highly subject to self-contamination;
  • Anticontamination with dUTP-UNG can be used in only one of the two steps;
  • The additional pipetting steps are time consuming and can result in sample mix up issues. (From:  Poritz et al., PLOS One Oct 19 (2011);   Purcell et al., J. Aq. An. Health 23:148 (2011);   Priyanka et al., Indian J Med Res. 144:327 (2016))