Nested PCR: Enhancing Sensitivity and Specificity

Nested polymerase chain reaction (PCR) is a two-stage amplification strategy that uses two sequential primer pairs to improve the detection of low-abundance nucleic acid targets. The first reaction amplifies an outer region of the template, and the second reaction uses primers positioned internal to the first pair to amplify a shorter product from the first reaction's output. This approach increases sensitivity by permitting a higher total number of amplification cycles and improves specificity because two separate primer sets must both bind to the same target sequence. Nested PCR is appropriate for diagnostic laboratories that need to detect pathogens present at very low concentrations, amplify specific members of polymorphic gene families, or work with degraded nucleic acid from formalin-fixed tissues. This article explains the scientific basis of nested PCR, compares it with alternative amplification methods, provides a practical protocol for implementation, and outlines the quality controls and contamination measures required for reliable results.

At a Glance

Nested PCR is a mature molecular technique with documented performance across veterinary and human diagnostic applications. The table below summarizes key characteristics that laboratory managers and technicians should consider when deciding whether to implement nested PCR in their diagnostic workflow.

The choice among these methods depends on the diagnostic question, sample type, available equipment, and the laboratory's capacity to manage contamination risk. Nested PCR offers a practical path to high sensitivity when real-time PCR instruments are unavailable or when the target sequence requires a longer amplicon for downstream characterization.

Scientific Basis of Nested PCR

Two-Stage Amplification Design

Nested PCR operates through two linked amplification reactions. The first reaction uses an outer primer pair that flanks a relatively long region of the target sequence. The product of this first reaction becomes the template for the second reaction, which uses an inner primer pair that anneals to sequences located within the first amplicon. The second reaction therefore amplifies a shorter, internal fragment. This design means that any product generated in the second reaction must have been recognized by both primer pairs, which substantially reduces the chance of amplifying nonspecific products. The use of two pairs of oligonucleotides allows a higher number of cycles to be performed, thereby increasing the sensitivity of the PCR. Nested PCR is an efficient method to amplify segments of long templates but requires knowledge of the sequence of the target [6].

Why Two Primer Pairs Improve Specificity

The specificity gain in nested PCR comes from the requirement that two independent primer-binding events occur on the same template molecule. In a single-round PCR, a primer pair may occasionally anneal to unintended but partially complementary sequences, producing spurious bands. In nested PCR, even if the outer primers generate some nonspecific products, the inner primers will only amplify those products that contain the internal target sequence. This double-selection process is the primary reason nested PCR can distinguish closely related sequences, such as individual members of a polymorphic gene family or specific pathogen strains within a mixed population [6].

Sensitivity Enhancement Through Additional Cycles

Each amplification round can be run for an optimal number of cycles without the plateau effects that limit single-round reactions. The first round typically runs 25 to 35 cycles, and the second round adds another 25 to 30 cycles. The total number of effective cycles is therefore higher than what is practical in a single reaction, allowing detection of templates present at very low copy numbers. Studies have demonstrated that nested PCR can detect pathogen DNA at concentrations far below the limit of conventional PCR. For example, a nested PCR targeting the HYR gene of the crab pathogen Metschnikowia bicuspidata achieved a detection limit of 6.10 × 10¹ copies per microliter, which was substantially more sensitive than PCR assays targeting ribosomal DNA regions [22]. Similarly, a modified nested PCR for cutaneous leishmaniasis detected as little as 2.55 femtograms of parasite DNA, compared with 25 femtograms for a single-round ITS1 PCR [20].

Applications in Veterinary and Diagnostic Settings

Detection of Low-Abundance Pathogens

Nested PCR is frequently used when clinical samples contain very few pathogen copies. In bovine herpesvirus-1 detection, a nested PCR assay identified the virus in semen samples with titres as low as 0.001 TCID50 per 50 microliters without requiring additional hybridization procedures. The assay proved more sensitive, faster, and easier to perform than standard viral isolation when tested on 101 semen samples from naturally infected bulls at an artificial insemination centre [11]. This example illustrates how nested PCR can replace time-consuming culture methods while improving detection rates.

Diagnosis of Parasitic Infections

Parasitic diseases often present diagnostic challenges because parasite numbers in clinical samples fluctuate and may be very low. A nested PCR for Plasmodium malariae targeting the 18S rRNA gene achieved a limit of detection of 0.5 parasites per microliter after optimization of cycling conditions and annealing temperature [16]. In the diagnosis of histomoniasis in poultry, adding a nested PCR step increased the sensitivity of detection compared with conventional PCR, although real-time PCR showed higher specificity [10]. For cutaneous leishmaniasis, a nested ITS1 PCR with novel inner primers detected more cases than slit skin smear microscopy, particularly in clinically doubtful lesions and atypical presentations [20].

Bacterial and Viral Detection in Complex Matrices

Nested PCR has proven valuable for detecting bacteria and viruses in samples that contain inhibitors or complex biological backgrounds. A nested PCR for Coxiella burnetii targeting the IS1111 repetitive element detected the pathogen in seven samples that were negative by conventional PCR, including spleen tissue from rodents and tick samples [23]. In wastewater surveillance, a semi-nested RT-PCR assay targeting the full spike protein region of SARS-CoV-2 successfully amplified fragments of approximately 2000 base pairs from samples with fewer than 10 copies per analyte, enabling variant identification even at very low RNA concentrations [14].

Genotyping and Variant Discrimination

The improved specificity of nested PCR makes it suitable for distinguishing closely related organisms or genetic variants. Dengue virus serotyping was accomplished by using consensus outer primers to amplify a 511-base pair product, followed by a nested PCR with type-specific primers that generated diagnostic fragment sizes for each of the four serotypes [7]. In hepatitis B virus drug resistance testing, nested PCR combined with pyrosequencing showed better consistency with predicted values than conventional PCR and was superior for detecting samples containing 90% mutant plasmid. The nested approach also demonstrated higher sensitivity than Sanger sequencing for clinical specimens with low viral loads, particularly those with HBV DNA at or below 10³ copies per milliliter [9].

Working with Degraded Nucleic Acid

Formalin-fixed paraffin-embedded tissues present a particular challenge for molecular diagnostics because formalin fixation causes DNA fragmentation. Nested PCR protocols have been developed specifically for such samples. Four nested PCR-RFLP approaches targeting CYP2D6 variants were optimized for archived breast cancer formalin-fixed paraffin-embedded tissues, demonstrating the feasibility of pharmacogenetic studies in resource-limited settings where fresh tissue is unavailable [15]. The two-stage design compensates for template degradation by first amplifying a longer region and then re-amplifying a shorter internal fragment from the first-round product.

Protocol for Implementing Nested PCR

Primer Design Considerations

Successful nested PCR begins with careful primer design. The outer primer pair should flank a region of 400 to 1000 base pairs, while the inner primers should anneal within this region and produce a final amplicon of 150 to 400 base pairs. The inner primers must be positioned internal to the outer primers, meaning their binding sites lie between the outer primer binding sites on the target sequence [6]. Several practical rules apply:

• Design both primer pairs against the same reference sequence to ensure the inner primers bind within the outer amplicon.
• Check that the inner primers do not overlap with the outer primer binding sites.
• Verify primer specificity using a sequence database search to avoid unintended annealing to related sequences.
• Aim for similar melting temperatures within each primer pair, typically 55 to 65 degrees Celsius.
• Avoid primer dimers and hairpin structures that reduce amplification efficiency.

For polymorphic targets, the outer primers may be designed as consensus primers that anneal to conserved regions across multiple variants, while the inner primers provide type-specific discrimination. This strategy was used effectively for dengue virus typing, where consensus primers amplified all four serotypes and type-specific inner primers generated diagnostic fragment sizes [7].

Reaction Setup and Cycling Conditions

The first-round reaction contains the outer primer pair, template DNA, polymerase, buffer, and nucleotides. After the first round is complete, a small aliquot of the product is transferred to a second reaction tube containing the inner primer pair and fresh reagents. The transfer volume is typically 1 to 5 microliters of first-round product into a 25 to 50 microliter second-round reaction.

Cycling conditions must be optimized for each primer pair. A study optimizing nested PCR for Plasmodium malariae evaluated cycle numbers of 25, 30, and 35 in the first round and 25 and 30 in the second round, along with annealing temperatures from 54 to 60 degrees Celsius for the inner primers. The optimal conditions were 35 cycles in the first round, 25 cycles in the second round, and an annealing temperature of 54 degrees Celsius, achieving a detection limit of 0.5 parasites per microliter [16]. This example demonstrates that cycle numbers and annealing temperatures should be empirically determined instead of assumed from published protocols.

Template Preparation and Quality Assessment

The quality of the template DNA or RNA directly affects nested PCR performance. For clinical specimens, extraction methods should remove inhibitors that can block polymerase activity. Blood, semen, feces, and tissue samples each require appropriate extraction protocols. In the bovine herpesvirus-1 study, filtering semen samples on chromatography columns before DNA extraction was essential for achieving high sensitivity [11]. For formalin-fixed tissues, specialized extraction methods that maximize recovery of fragmented DNA are necessary [15].

Before running nested PCR, assess template quality and quantity. Spectrophotometric measurement of DNA concentration and purity ratios provides a preliminary check. For degraded samples, amplification of a housekeeping gene can confirm that the template is amplifiable. If the control gene fails to amplify, the sample may contain inhibitors or the DNA may be too degraded for reliable testing.

Detection and Analysis of Products

After the second round, amplification products are separated by agarose gel electrophoresis and visualized with a DNA-binding dye. The expected fragment size is determined by the inner primer positions. A single clear band of the expected size indicates a positive result. Silver-stained polyacrylamide gels offer higher resolution and sensitivity than agarose gels and were used in the bovine herpesvirus-1 assay to detect low-level amplification products [11].

For definitive identification, the amplified product can be sequenced or subjected to restriction fragment length polymorphism analysis. Nested PCR products are well suited for direct sequencing because the two-stage amplification generates sufficient template for downstream analysis [23]. In the Cryptosporidium diagnostic workflow, a nested PCR detects all Cryptosporidium species, and the amplicons are then differentiated by sequencing [13].

Quality Controls and Contamination Prevention

The Central Risk: Carryover Contamination

The most significant limitation of nested PCR is the elevated risk of carryover contamination. Because the first-round product must be transferred to a second reaction tube, any aerosol or droplet containing amplified DNA can contaminate subsequent reactions. The second-round primers will amplify the first-round product with high efficiency, so even trace amounts of carryover can produce false positives. This risk is well documented, and studies have noted that previously developed nested PCR methods tend to have low specificities due to high false-positive rates [8].

Physical Separation of Workflow Stages

Contamination control begins with physical separation of the workflow. Ideally, the laboratory should have dedicated areas for reagent preparation, sample processing, first-round amplification, and second-round setup. The second-round setup should occur in a clean area that never receives clinical samples or first-round products. Separate pipettes, tube racks, and laboratory coats should be assigned to each area. The World Health Organization Laboratory Quality Management System Handbook provides general guidance on organizing laboratory workflows to minimize contamination and ensure reliable results [1].

One-Tube Nested PCR as a Contamination-Reduction Strategy

To address the contamination risk inherent in two-step nested PCR, one-tube formats have been developed. A one-tube nested PCR for Mycobacterium tuberculosis detection in cerebrospinal fluid maintained the sensitivity of conventional two-step nested PCR while reducing both the chance of cross-contamination and the time required for analysis. The assay achieved a limit of detection of 1 femtogram of M. tuberculosis DNA and showed 89% overall sensitivity and 100% specificity for tuberculous meningitis patients [24]. Laboratories concerned about contamination should evaluate whether a one-tube format is available for their target of interest.

Controls to Include in Every Run

Every nested PCR run should include appropriate controls to validate the results:

• A no-template control for each primer pair to detect reagent contamination.
• A positive control with a known amount of target DNA to confirm amplification efficiency.
• A negative extraction control processed alongside clinical samples to detect contamination during extraction.
• For RNA targets, a reverse transcription control to confirm successful cDNA synthesis.

The World Health Organization Laboratory Quality Management System Handbook emphasizes that quality control samples must be processed in the same manner as patient samples to provide meaningful monitoring of the entire testing process [1].

Validation of Assay Performance

Before implementing a nested PCR assay for diagnostic use, the laboratory should validate its performance characteristics. Sensitivity and specificity should be established using well-characterized samples. The U.S. Food and Drug Administration Bioanalytical Method Validation Guidance describes the expectations for validating analytical methods, including assessment of accuracy, precision, selectivity, and sensitivity [4]. The National Center for Advancing Translational Sciences Assay Guidance Manual provides additional detail on assay development and validation approaches [3].

Validation should include determination of the limit of detection using serial dilutions of a standard template. A study of nested PCR for Coxiella burnetii used tenfold serial dilutions to establish the detection limit and demonstrated that the nested assay detected ten times less DNA than the conventional PCR [23]. For quantitative applications, linearity should be assessed across the expected range of template concentrations [9].

Comparison with Alternative Amplification Methods

Nested PCR versus Real-Time PCR

Real-time PCR offers several advantages over nested PCR, particularly in specificity and contamination control. In a study comparing assays for Pneumocystis jiroveci detection, both nested and real-time PCR showed high sensitivity ranging from 62.5% to 100% depending on specimen type. However, in a subset of 71 confirmed cases and 70 negative cases, the sensitivity and specificity were 94% and 81% for nested PCR and 94% and 96% for real-time PCR, respectively. Real-time PCR had statistically significantly better specificity than nested PCR and was likely to generate fewer false positives [8]. Similarly, in histomoniasis diagnosis, real-time PCR was more specific than nested PCR, although conventional PCR was more sensitive than real-time PCR and nested PCR further increased sensitivity [10].

The closed-tube format of real-time PCR eliminates the product transfer step that creates contamination risk in nested PCR. Real-time PCR also provides quantitative data across a wide dynamic range, which is valuable for monitoring treatment response or estimating pathogen load. However, real-time PCR requires specialized instrumentation and reagents that may not be available in all laboratories.

Nested PCR versus Conventional PCR

Nested PCR consistently outperforms conventional single-round PCR in sensitivity. In the detection of Coxiella burnetii, seven samples that were negative by conventional PCR were positive by nested PCR [23]. For hepatitis B virus drug resistance testing, nested PCR pyrosequencing showed higher sensitivity than conventional PCR pyrosequencing, particularly for samples containing low proportions of mutant virus [9]. The sensitivity advantage of nested PCR is most pronounced when target concentrations are very low, such as in early infection or in samples with inhibitory substances.

Nested PCR versus Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) has emerged as an alternative to PCR-based methods. In a comparative study using Entamoeba histolytica DNA from fecal samples, LAMP achieved a limit of detection of one trophozoite, while both qPCR and nested PCR detected 100 trophozoites and conventional PCR detected 1000 trophozoites. LAMP also required less amplification time [21]. However, LAMP requires specific primer sets and may be less flexible for downstream sequencing applications. The choice between LAMP and nested PCR depends on the laboratory's needs for quantification, sequencing, and throughput.

Common Failure Patterns and Troubleshooting

No Amplification in the Second Round

When the second-round reaction produces no visible product, several causes should be investigated. The first-round amplification may have failed due to poor template quality, inhibitors, or suboptimal cycling conditions. Run the first-round product on a gel to confirm that the outer primers produced the expected fragment. If the first round failed, the problem lies in template preparation or outer primer performance. If the first round succeeded but the second round failed, the inner primers may not anneal efficiently under the chosen conditions, or the transfer volume may have been too small.

Multiple Bands or Smearing

Multiple bands in the second-round product indicate nonspecific amplification. The inner primers may be annealing to unintended sequences, or the annealing temperature may be too low. Increasing the annealing temperature in 2-degree increments can improve specificity. Reducing the number of cycles in either round may also reduce nonspecific products. The Plasmodium malariae optimization study demonstrated that annealing temperature had a significant effect on primer specificity, with the MAL1/MAL2 primer pair showing no amplification of other Plasmodium species at the optimized temperature [16].

False Positives from Contamination

False positives are the most serious failure mode in nested PCR because they can lead to incorrect clinical diagnoses. If negative controls show amplification, the assay is contaminated. The contamination source may be first-round products, positive control DNA, or environmental DNA. Decontaminate all surfaces and equipment, replace reagents, and review the workflow to identify where contamination entered. The World Health Organization Laboratory Biosafety Manual provides guidance on safe handling of amplified products and decontamination procedures [2].

Inconsistent Results Between Runs

Run-to-run variability can arise from differences in reagent lots, thermal cycler performance, or operator technique. Standardize reagent preparation and storage, calibrate thermal cyclers regularly, and ensure that all operators follow the same protocol. The National Center for Advancing Translational Sciences Assay Guidance Manual recommends documenting all assay parameters and monitoring performance over time to detect drift [3].

Records and Documentation

What to Record for Each Run

Accurate documentation is essential for interpreting nested PCR results and for troubleshooting when problems arise. For each run, record:

• Sample identification and source
• DNA or RNA extraction method and date
• Template concentration and quality metrics
• Primer lot numbers and working concentrations
• Master mix composition and reagent lot numbers
• Thermal cycling parameters for both rounds
• Transfer volume from first to second round
• Gel electrophoresis conditions and image
• Control results and any anomalies

The World Health Organization Laboratory Quality Management System Handbook emphasizes that documentation must be sufficient to allow reconstruction of the testing process and to support the validity of reported results [1].

Maintaining a Contamination Log

A dedicated log for contamination events helps identify patterns and sources. Record any unexpected amplification in negative controls, the date and time of the event, the operator, and the corrective actions taken. Review this log regularly to assess whether contamination control measures are effective.

Linking Results to Clinical or Management Decisions

Nested PCR results should be interpreted in the context of the clinical question and the limitations of the assay. A positive result in a nested PCR does not necessarily indicate active infection, as the high sensitivity may detect nonviable organisms or low-level carriage. In the Pneumocystis jiroveci study, the lower specificity of nested PCR compared with real-time PCR was attributed to false positives, highlighting the need for careful interpretation of positive results in patients without compatible clinical findings [8].

Biosafety Considerations

Handling of Clinical Specimens

All clinical specimens should be handled according to standard biosafety precautions. The World Health Organization Laboratory Biosafety Manual provides comprehensive guidance on risk assessment, containment levels, and safe practices for laboratories working with infectious materials [2]. Specimens that may contain highly pathogenic organisms require additional precautions, including appropriate personal protective equipment and containment facilities.

Safe Handling of Amplified Products

Amplified DNA products are not infectious, but they represent a contamination hazard for the laboratory. First-round products should be opened only in the designated amplification area, and all tubes containing amplified DNA should be disposed of according to local regulations. Never open first-round tubes in the reagent preparation or sample processing areas.

Waste Disposal

Agarose gels containing DNA-binding dyes and used reaction tubes should be collected and disposed of as laboratory waste. Follow institutional and local regulations for disposal of molecular biology waste. The Laboratory Biosafety Manual provides general principles for waste management in biomedical laboratories [2].

Limitations and Interpretation Caveats

Sequence Knowledge Requirement

Nested PCR requires knowledge of the target sequence to design both primer pairs. This requirement limits the application of nested PCR to organisms or genes with known sequences. For novel or uncharacterized pathogens, other approaches such as metagenomic sequencing may be more appropriate [6].

Semiquantitative Nature

Nested PCR is primarily a qualitative or semiquantitative method. The endpoint analysis does not provide accurate quantification across a wide dynamic range. If quantitative results are needed, real-time PCR is the preferred method. The hepatitis B virus drug resistance study demonstrated that nested PCR combined with pyrosequencing could provide quantitative information about mutant proportions, but this required additional instrumentation and analysis [9].

Specificity Limitations in Certain Applications

While nested PCR generally improves specificity, it does not eliminate false positives. The Pneumocystis jiroveci study found that nested PCR had a specificity of 81% compared with 96% for real-time PCR, indicating that the nested approach still produced false positives in a subset of samples [8]. Laboratories should be aware that high sensitivity can come at the cost of reduced clinical specificity, particularly in populations with low disease prevalence.

Time and Labor Requirements

Nested PCR requires more hands-on time than single-round PCR because of the two amplification steps and two gel runs. The one-tube nested PCR format reduces manipulation time and contamination risk while maintaining sensitivity [24]. Laboratories with high sample volumes should consider whether the increased sensitivity justifies the additional labor.

Professional Escalation Criteria

Laboratory personnel should escalate results or technical problems to a supervisor or referring clinician under specific circumstances:

• A positive nested PCR result in a patient with no compatible clinical findings, which may represent contamination or false positivity.
• Repeated contamination events that cannot be traced to a specific source.
• Unexplained loss of assay sensitivity, indicated by failure of positive controls.
• Results that will guide treatment decisions for serious infections, where confirmation by an independent method may be warranted.
• Detection of organisms with public health implications, such as notifiable diseases, which require reporting to the appropriate authorities.

The World Health Organization Laboratory Quality Management System Handbook advises that laboratories must have procedures for reporting results to clinicians and for managing critical or unexpected findings [1].

Frequently Asked Questions

What is the main advantage of nested PCR over conventional PCR?

Nested PCR provides higher sensitivity and specificity than conventional single-round PCR. The two-stage design allows more total amplification cycles, which increases the ability to detect low-abundance targets. The requirement that two separate primer pairs bind to the same template reduces nonspecific amplification. Studies have demonstrated that nested PCR detects pathogens in samples that are negative by conventional PCR, such as Coxiella burnetii in rodent spleen and tick samples [23].

Why does nested PCR have a higher contamination risk?

Nested PCR requires transferring first-round product to a second reaction tube. This transfer step creates opportunities for aerosol or droplet contamination of other reactions. Because the second-round primers amplify the first-round product very efficiently, even trace amounts of carryover can produce false positives. One-tube nested PCR formats reduce this risk by keeping both amplification steps in a single closed tube [24].

When should I choose nested PCR instead of real-time PCR?

Choose nested PCR when real-time PCR instrumentation is unavailable or when the target region needs to be longer than what real-time PCR can efficiently amplify. Nested PCR is also useful when the laboratory needs to generate amplicons for sequencing or restriction fragment length polymorphism analysis. However, real-time PCR offers better specificity and lower contamination risk, so it is preferred when quantitative results are needed or when the laboratory can support the instrument cost [8].

How many cycles should I use in each round of nested PCR?

Cycle numbers must be optimized for each assay. A study optimizing nested PCR for Plasmodium malariae found that 35 cycles in the first round and 25 cycles in the second round with an annealing temperature of 54 degrees Celsius provided optimal performance [16]. Start with published protocols for similar targets and adjust cycle numbers based on the intensity of the expected bands and the presence of nonspecific products.

Can nested PCR be used with formalin-fixed paraffin-embedded tissues?

Yes, nested PCR is well suited for formalin-fixed paraffin-embedded tissues because the two-stage design compensates for DNA fragmentation caused by formalin fixation. Protocols have been developed for pharmacogenetic testing using archived breast cancer tissues, demonstrating the feasibility of this approach in resource-limited settings [15].

What controls are essential for a nested PCR run?

Essential controls include a no-template control for each primer pair, a positive control with known target DNA, and a negative extraction control processed alongside clinical samples. These controls detect reagent contamination, confirm amplification efficiency, and monitor the extraction process. The World Health Organization Laboratory Quality Management System Handbook emphasizes that quality control samples must be processed in the same manner as patient samples [1].

How do I confirm that my nested PCR product is the correct target?

Confirm the identity of the amplified product by sequencing or restriction fragment length polymorphism analysis. Nested PCR products are well suited for direct sequencing because the two-stage amplification generates sufficient template [23]. In the Cryptosporidium diagnostic workflow, nested PCR amplicons are sequenced to differentiate species [13].

What should I do if my negative controls show amplification?

Amplification in negative controls indicates contamination. Immediately stop testing, decontaminate all surfaces and equipment, replace reagents, and review the workflow to identify the contamination source. Document the event in a contamination log and implement corrective actions before resuming testing. The World Health Organization Laboratory Biosafety Manual provides guidance on decontamination procedures [2].

Related Diagnostic Guides

• Site-Directed Mutagenesis Using Overlap Extension PCR: Protocol and Primer Design
• Nested PCR: Principles, Protocol, and Applications
• PCR Primer Design: Rules, Tools, and Validation
• Contamination Control in PCR and qPCR: Sources, Prevention, and Decontamination
• Spread Plate Method: Advantages, Disadvantages, and Step-by-Step Protocol

References and Further Reading

• Laboratory Quality Management System Handbook. World Health Organization.
• Laboratory Biosafety Manual. World Health Organization.
• Assay Guidance Manual. National Center for Advancing Translational Sciences.
• Bioanalytical Method Validation Guidance. U.S. Food and Drug Administration.
• NCBI Literature Resources. National Center for Biotechnology Information.
• Nested Polymerase Chain Reaction (PCR).. Cold Spring Harbor protocols, 2019.
• Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.. Journal of clinical microbiology, 1992.
• Sensitivity and specificity of nested and real-time PCR for the detection of Pneumocystis jiroveci in clinical specimens.. Diagnostic microbiology and infectious disease, 2006.
• [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].. Nan fang yi ke da xue xue bao = Journal of Southern Medical University, 2012.
• Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the diagnosis of histomoniasis.. Avian diseases, 2005.
• A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls.. Veterinary microbiology, 1998.
• Improved PCR/nested PCR approaches with increased sensitivity and specificity for the detection of pathogens in hard ticks.. Ticks and tick-borne diseases, 2013.
• Cryptosporidium Diagnostic Assays: Molecular Detection.. Methods in molecular biology (Clifton, N.J.), 2020.
• Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.. 2026.
• Technical implementation for african pharmacogenetic studies on &lt,i&gt,CYP2D6&lt,/i&gt, from archived breast cancer formalin fixed paraffin-embedded (FFPE) tissues.. 2026.
• Optimization of Nested-PCR for the detection of &lt,i&gt,Plasmodium malariae&lt,/i&gt, in laboratory settings in Southern Vietnam.. 2026.
• Rapid identification of antigen-specific TCRs for cancer immunotherapy.. 2026.
• Enterovirus Testing in Hand, Foot, and Mouth Disease and Herpangina: A Highly Sensitive Single-Round VP4-VP2 Reverse-Transcription Polymerase Chain Reaction Assay with a Redesigned Reverse Primer.. 2026.
• DNA from Slow-Growing Mycobacteria in Culture Negative Sputa Reveal Common Exposure to Mycobacteria.. 2026.
• Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka. Microorganisms, 2022.
• Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample. BMC Biotechnology, 2020.
• Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis. Frontiers in Cellular and Infection Microbiology, 2022.
• Clinical and epidemiological use of nested PCR targeting the repetitive element IS1111 associated with the transposase gene from Coxiella burnetii. Brazilian Journal of Microbiology, 2017.
• Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis. PLoS ONE, 2017.
• [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].. Nan Fang Yi Ke Da Xue Xue Bao Journal of Southern Medical University, 2012.
• Sensitivity and specificity of nested PCR and spoligotyping as quick diagnostic tests for bovine tuberculosis in fresh tissue. Tecnica Pecuaria En Mexico, 2010.
• Sensitivity and specificity of nested PCR and spoligotyping as quick diagnostic tests for bovine tuberculosis in fresh tissue. Revista Mexicana De Ciencias Pecuarias, 2010.
• Sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi Chinese Journal of Parasitology Parasitic Diseases, 2007.

This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.