Nested PCR is a modification of PCR designed to increase the sensitivity and specificity of the assay reaction. It involves the use of two primer sets directed against the same target and two successive PCR reactions.
The first set of primers is designed to anneal to sequences upstream from the second set of primers, whereas the second set of primers is situated internally or nested with respect to the first set of primers. The first set of primers also called “outer primers” amplify a large fragment of the gene which is used as a template in the second round of PCR that targets a smaller region of the amplicon using the second set of primers also known as “inner primers or nested primers.”
The traditional approach to nested PCR was to perform a number of PCR cycles using the first set of primers, and then open the reaction vessel and add the second, nested, set of primers to run the second PCR cycle. The major problem with this approach is amplicon contamination in the laboratory and a consequential loss of specificity of the assay. To address this issue single-tube nested PCR (STNPCR) reactions have been developed, wherein both sets of primers are added to the initial reaction vessel and an extended PCR is performed.
Amplicons from this PCR assays are visualized by electrophoresing the reaction mixture in 2% ethidium bromide-stained agarose gel along with a molecular weight marker.
Nested PCRs are sometimes necessary to compensate for inefficient first-round PCR due to primer mismatches so, if we can use well-matched primers for first-round PCR nested approach may not be needed in many circumstances.
Nested PCR reduces the nonspecific amplification of the target sequence.
This is because nested primers will not find priming sites on any primer dimers or nonspecific artifacts generated in the primary PCR. Nested primers will only prime any specific product generated in the primary PCR, thus maintaining PCR specificity.
Applications of Nested PCR
To improve the sensitivity of the assay, nested PCR has been used in many PCR assays. It is particularly useful for suboptimal nucleic acid samples, such as those extracted from formalin-fixed, paraffin-embedded tissue
Nested PCRs have proven valuable for the detection of microorganisms when they are present in very low quantities. For example:
- detection of Rickettsia, Bartonella, and similar organisms in the blood (bacteremia) and tissues,
- detection of herpesvirus and enterovirus in the CSF, and
- detection of M. tuberculosis in a sputum sample.
The BioFire FilmArray from bioMérieux is a commercially available system that employs nested, multiplex, and singleplex PCR reactions for the detection of a variety of pathogens.
- Susceptible to contamination: The extreme sensitivity of nested PCR comes with its own set of problems. This test is highly susceptible to contamination as it involves more time for sample manipulation. Contamination mostly occurs during the transfer of the first-round product to the second tube for the second round of amplification.
- Costly: This PCR assay is also more costly as it involves the use of two separate reactions to arrive at one result. The cost will increase dramatically if assays are repeated when contamination occurs.
References and further reading
- Chang-Hui Shen (2019). Amplification of Nucleic Acids. Diagnostic Molecular Biology.
- Deepachandi, B., Weerasinghe, S., Soysa, P. et al (2019). A highly sensitive modified nested PCR to enhance case detection in leishmaniasis. BMC Infect Dis 19, 623 doi:10.1186/s12879-019-4180-3
- Souza G., Almeida A., Farias A., Leal N., Abath F. (2007). Development and Evaluation of a Single Tube Nested PCR Based Approach (STNPCR) for the Diagnosis of Plague. In: Perry R.D., Fetherston J.D. (eds) The Genus Yersinia. Advances In Experimental Medicine And Biology, vol 603. Springer, New York, NY