​​Additional References - Needs/Advantage of Multiplex and Nested PCR
1 The primary advantage of Multiplex PCR is obtaining more information out of a single reaction. This is particularly important when the sample input is limited. 

2 By targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would require several times the reagents (including the sample) and more time to perform.

3 The primary disadvantage of all PCR methods is the chance of false positive signals due to primer binding to non-specific DNA sequences… The primary advantage of nested PCR is specificity…. If the first primers amplify an unwanted DNA sequence, it is very unlikely that the second set of primers the same unwanted region. Nested PCR can increase the yield and specificity of amplification of the target DNA....Nested PCR requires two sequential PCR steps… The products of the first step need to be diluted (or cleaned up or treated) and used in the second PCR step… This creates a serious PCR product contamination risk as well as additional pipetting steps, which can result in sample mix up issues.

4 Nested PCR is used to increase the specificity of DNA amplification.
5 Nested PCR is a variation of standard PCR that enhances the specificity and yield of the desired amplicons.

6 Nested PCR increases sensitivity over conventional PCR… Specificity of nested PCR is similar to that of probe-based assays… Although the use of nested primer sets was described very early in the history of PCR it has not been widely deployed in clinical settings because, in most systems, it is an open-tube procedure that is highly subject to self-contamination.

7 The disadvantage of the nested PCR is that no anticontamination with dUTP-UNG can be used. Therefore, it is most sensitive to contamination.”
8 A serious disadvantage of nested PCRs is the high risk of carryover contamination. In our study omp1-based nested PCR was found to be useful only as a confirmatory test but not for routine testing of specimens. Furthermore, UNG, which destroys products from previous amplifications, can be used in a nested PCR only in the second round of amplification.
9 On the other hand, the use of nested PCR increases the risk of contamination of samples, which may lead to a more frequent appearance of false positive results. “