Nested PCR: Principle and Applications

A 45-year-old farmer from the Bihar region of India presents with two weeks of fever, headache, and a eschar — a dark, crusted skin lesion at the site of a mite bite on his forearm. The clinician suspects scrub typhus caused by Orientia tsutsugamushi. Blood is drawn and sent to the reference laboratory.

Standard PCR on the blood sample returns negative. The organism is present — the clinical picture is clear — but the bacterial load in peripheral blood in scrub typhus is extremely low, often just a few copies per milliliter. Standard PCR lacks the sensitivity to detect it reliably at that concentration.

The laboratory repeats the test using nested PCR. The outer primer pair amplifies a large fragment of the Orientia 47-kDa gene. The product of that first reaction becomes the template for the second reaction, where inner primers amplify a smaller, specific region nested within the first amplicon. The signal is amplified twice over. This time, the result is positive.

Nested PCR did not just improve sensitivity here — it made the diagnosis possible at all. In infections where organisms circulate in blood at very low copy numbers, nested PCR is often the only PCR method capable of detection.

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.

Figure: Nested PCR (Image Source:https://www.thermofisher.com/)

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.

Figure: Nested Polymerase Chain Reaction (PCR)

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.

Advantage

It 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

To improve the sensitivity of the assay, it 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.

Commercial application

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.

For an overview of all PCR types used in clinical microbiology, see Polymerase Chain Reaction: Steps, Types, and Applications

Limitations

• Susceptible to contamination: The extreme sensitivity of this type of 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.

How to Remember

Nested PCR = two rounds of amplification, each inside the previous. The name "nested" is the memory device itself. The inner primers sit inside the sequence amplified by the outer primers — like a smaller box nested inside a larger one. Round 1 (outer primers) amplifies a large region. Round 2 (inner primers) amplifies a smaller region within that product. Two rounds of amplification mean exponentially more copies of the target — and two rounds of primer specificity mean far fewer non-specific products.

Outer primers = big net; inner primers = fine sieve. The outer primers cast a wide net over the target region, amplifying a large fragment — even from a degraded or low-copy template. The inner primers then act as a fine sieve, amplifying only the specific sequence of interest from within that large fragment. Anything non-specific that slipped through the first net is unlikely to contain the inner primer binding sites and is eliminated in the second round.

High sensitivity comes with high contamination risk — the trade-off. Nested PCR is the most sensitive PCR method for low-copy targets — and also the most contamination-prone, because the first-round product is opened and transferred to a second tube in traditional two-tube nested PCR. Even nanogram quantities of that first-round amplicon contaminating the laboratory environment can seed false positives in subsequent runs. This is why single-tube nested PCR (STNPCR) was developed — both primer sets added at the start, no tube opening between rounds.

Clinical trigger for nested PCR: "present in very low quantities." Whenever a question describes an organism present in very low copy numbers — Rickettsia, Bartonella, M. tuberculosis in paucibacillary samples, viruses in CSF, Leishmania in tissue — nested PCR is the answer. The exam pattern is: low pathogen load + need for high sensitivity = nested PCR.

Key exam facts in one table

References and further reading

1. Chang-Hui Shen (2019). Amplification of Nucleic Acids. Diagnostic Molecular Biology. Academic Press. https://doi.org/10.1016/B978-0-12-802823-0.00009-2
2. Deepachandi, B., Weerasinghe, S., Soysa, P. et al (2019). A highly sensitive modified nested PCR to enhance case detection in leishmaniasis. BMC Infectious Diseases 19, 623. https://doi.org/10.1186/s12879-019-4180-3
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.