Polymerase Chain Reaction (PCR)

Testing Procedures

PCR begins with the extraction of a small nucleic acid sample, typically DNA or RNA, into a reaction tube. The process consists of three major phases: denaturation, annealing, and extension. During denaturation, DNA is heated to 95°C to disrupt hydrogen bonds between complementary base pairs of the double-stranded molecule. Annealing follows immediately, cooling the denatured DNA to a temperature typically ranging from 55°C to 72°C, allowing primers to bind to their complementary sequences. Annealing is most effective at 55°C to 72°C.

The optimal annealing temperature depends on the physical and chemical properties of the primers in solution. Primers are typically 20 to 25 nucleotides long. During annealing, primers bind to complementary sequences on single-stranded DNA by pairing their 3′ ends to the template strand, providing a starting point for DNA synthesis. This interaction enables the subsequent synthesis of double-stranded DNA during the extension phase. The final phase uses a temperature of 75°C to 80°C to optimize DNA polymerase activity and promote strand elongation.

DNA polymerase requires a primer annealed to single-stranded DNA to initiate strand synthesis. The enzyme synthesizes new strands in the 5′ to 3′ direction, generating sequences complementary to the template strands. This process is repeated using a thermal cycler, a device that regulates the temperature and duration of each step in the cycle. Repeated cycling results in the amplification of multiple copies of the target DNA within the tube. Amplification efficiency declines after 30 to 40 cycles due to reagent depletion, accumulation of byproducts, reduced enzyme activity, and product-template competition, including accumulation of pyrophosphate molecules, excessive self-annealing, and the presence of PCR inhibitors. Inhibitors that interfere with PCR include proteinase K, phenol, and EDTA.

Proteinase K can inhibit PCR by degrading DNA polymerase and other essential proteins if not adequately removed during sample preparation. Other substances that negatively affect PCR include ionic detergents, heparin, spermidine, and hemoglobin. Bromophenol blue and xylene cyanol may also disrupt PCR reactions. To minimize these effects, DNA templates may be purified by dialysis or ethanol precipitation. Additional purification methods include chloroform extraction and chromatography.[5]

After PCR amplification, agarose gel electrophoresis with ethidium bromide staining is typically performed to visualize DNA. The gel is examined under ultraviolet light. To verify result specificity and reduce nonspecific amplification, such as primer dimers, DNA may be transferred to a membrane and probed, as in Southern blot hybridization.

PCR offers several advantages in basic and biomedical sciences. This modality has become the gold standard for numerous applications due to its speed, sensitivity, and reliability. Results are typically obtained within a few hours, though extended workflows may take up to 3 days. A minimal input of 1 to 100 ng of DNA or RNA is often sufficient to initiate the reaction, though some highly sensitive assays can use even lower amounts. PCR can amplify 10⁶ to 10⁹ copies of DNA within a short time. The incorporation of restriction sites at terminal ends enables efficient downstream cloning and expression of amplification products.

Real-time Polymerase Chain Reaction

Real-time PCR is a widely used method for analyzing small DNA segments, offering advantages such as elimination of post-PCR processing, use of fluorescent dyes or probes, and real-time monitoring of amplicon formation. The primary distinction between real-time and conventional PCR is that real-time PCR allows immediate detection of amplified products during the reaction, rather than after amplification is complete. This process is accomplished through the incorporation of fluorescent molecules, either intercalating dyes or sequence-specific probes, that emit signals proportional to DNA accumulation.

Despite these advantages, real-time PCR is more expensive than conventional PCR due to its specialized instrumentation and reagents. Additionally, some fluorescent dyes are not universally compatible with all platforms, owing to hardware limitations, such as excitation and emission wavelength incompatibility or insufficient filter sets.[6]

Reverse Transcription Polymerase Chain Reaction

Reverse transcription PCR (RT-PCR) uses messenger RNA as a template for DNA amplification by reverse transcriptase, often derived from retroviruses, to generate complementary DNA (cDNA). RT-PCR is often combined with conventional PCR to assess specific gene expression qualitatively.

RT-PCR and real-time PCR may be used together to evaluate quantitative differences in gene expression across multiple samples. During the COVID-19 pandemic, RT-PCR served as the primary diagnostic method due to its high sensitivity, specificity, and rapid turnaround time. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specimens are typically obtained from the upper respiratory tract.

Sample collection sites include the nasopharynx, oropharynx, nostrils, and oral cavity. Specimens are collected using swabs, washes, or bronchoalveolar lavage.[7]

Interfering Factors

Several limitations affect the accuracy and reliability of PCR. Extreme sensitivity allows detection of even minimal contamination in DNA or RNA samples, which may produce inaccurate results. Primer design requires precise sequence targeting of specific pathogens or genes. Occasional nonspecific annealing to closely related but unintended sequences can interfere with amplification specificity. Primer-dimer formation, when amplified by DNA polymerase, introduces competition for PCR reagents and reduces overall efficiency.

Results, Reporting, and Critical Findings

In PCR, DNA amplification may be monitored using fluorescent dyes that bind to double-stranded DNA or with sequence-specific probes. The amplification process includes a quantification cycle, defined as the number of fractional cycles required for fluorescence to reach a measurable threshold.

After determining the quantification cycle, either qualitative conclusions may be drawn or quantitative analyses may proceed. Quantification cycle (Cq) values depend on PCR efficiency, which refers to the fold increase in product per cycle. Efficiency ranges from 1 to 2, with a fold value of 2 representing 100% efficiency. PCR efficiency is assessed using standard curves and individual amplification curves.

Standard curve–based assessments are prone to dilution errors, potentially affecting quantification accuracy in clinical and biological samples. In contrast, amplification curve analysis excludes such confounding variables but may yield results that differ from those obtained using standard curves. Accurate quantification of the target depends on reliable amplification efficiency, which directly influences interpretation.

Low PCR efficiency requires additional cycles to reach the quantification threshold, resulting in a higher quantification cycle value. When a valid probe-based assay is used, the presence of amplification indicates that the sample contains the target sequence, supporting a positive diagnosis. However, the absence of amplification is not a reliable criterion for classifying a reaction as negative due to Poisson variation.

Quantitative PCR (qPCR) measures DNA or RNA in diagnostic and biological samples by analyzing the quantification cycle. Many qPCR analyses assume 100% amplification efficiency. Reporting may involve quantification cycle values, difference in quantification cycle (Δ Cq) values, or difference in the difference in quantification cycle (ΔΔ Cq) values. Applying efficiency correction is essential to accurately interpret qPCR results across biological, clinical, and diagnostic settings. Consideration of these parameters improves the reliability of qPCR-based analyses.[8]

The stage and severity of a patient’s disease may be assessed by interpreting quantification cycle values in conjunction with clinical presentation and medical history. Serial PCR testing allows clinicians to track disease progression and evaluate recovery by comparing changes in quantification cycle values over time. These values can also assist contact tracers in identifying individuals with higher viral genomic loads, suggesting a greater risk of transmission.[9]

Clinical Significance

PCR is widely used in basic and biomedical sciences due to its high sensitivity, specificity, and rapid processing time, making it valuable in both laboratory and clinical settings.[10] This technique is frequently employed to detect viral pathogens, including human papillomavirus, HIV, herpes simplex virus, severe acute respiratory syndrome coronavirus 2, varicella-zoster virus, enterovirus, cytomegalovirus, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.[11] The presence of bacterial, fungal, and parasitic organisms, as well as various immunodeficiencies, may be identified through PCR, making it an essential tool in clinical diagnoses and practice.

The rapid identification of microbial pathogens through real-time PCR enables clinicians to provide timely, targeted treatment, thereby reducing hospitalizations and preventing inappropriate antibiotic use and, in turn, antibiotic resistance. Real-time PCR can detect specific bacterial species, such as Mycobacterium species, Leptospira genospecies, Chlamydia species, Legionella pneumophila, Listeria monocytogenes, and Neisseria meningitidis. Real-time PCR has also proven effective in detecting and analyzing antibiotic-resistant strains, including Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, and Enterococcus.

In addition, fulminant diseases are detected and evaluated early due to the high sensitivity, specificity, and speed of real-time PCR. Thus, this modality is optimal for medical conditions such as meningitis, sepsis, and inflammatory bowel diseases.

Additional microbial pathogens associated with foodborne illness, including group B Streptococci, Mycobacterium species, Bacteroides vulgatus, and Escherichia coli, may be identified through real-time PCR testing. The rapid turnaround of real-time PCR facilitates early detection, supports source tracing, and aids in controlling ongoing and potential outbreaks. Fungal, parasitic, and protozoan organisms such as Aspergillus fumigatus, Aspergillus flavus, Cryptosporidium parvum, and Toxoplasma gondii have also been detected using this method.

PCR is further employed to investigate the histopathology of viral and cellular genes for the diagnosis and understanding of malignant human diseases. Applications of PCR extend to forensic analysis, point mutation detection, DNA sequencing, and in vitro mutagenesis. This technique efficiently screens and identifies specific alleles, making it suitable for prenatal genetic testing for carrier status. PCR can also detect disease-associated mutations both in utero and in adult samples.[12]

Quality Control and Lab Safety

Conventional PCR is considered the gold standard for screening and detection across a wide range of scientific applications due to its reliable results. Proper handling after amplification is essential to ensure accurate assessment of the amplicon. Inadequate postprocedural practices in conventional PCR may lead to uncontrolled dissemination of amplified DNA within the laboratory.

To minimize contamination, PCR must be performed in a designated area of the laboratory isolated from general workspace activity. Limiting airflow and movement in this space is critical. Personal protective equipment, including face masks, gloves, and hair covers, must be worn consistently. All solutions must be prepared and stored using uncontaminated equipment such as pipettes, glassware, and plasticware that have not been exposed to DNA.

Enzymes and buffers should be stored in a designated section of the freezer located near the laminar flow hood. Reagents should be discarded immediately after use. PCR procedures should be conducted within a laminar flow hood equipped with ultraviolet lighting. Essential equipment such as pipettes, sterile gloves, and a microcentrifuge must be available within this workspace.

Automatic pipettes increase the risk of cross-contamination and should be replaced with positive displacement pipettes for accurate reagent handling. Disposable laboratory materials, including pipette tips and tubes, must not be autoclaved prior to use and should be handled directly from sterile packaging.

Microcentrifuge tubes containing PCR reagents should be centrifuged for approximately 10 seconds before use to ensure that the contents settle at the bottom, thereby reducing the risk of aerosol contamination. Postamplification procedures must be conducted at a separate laboratory bench, away from the PCR-dedicated area.

Enhancing Healthcare Team Outcomes

Efficient use of PCR by the interprofessional healthcare team facilitates early detection of bacterial and viral pathogens, enabling earlier initiation of treatment. This approach also contributes to reducing antibiotic resistance and limiting the spread of viral outbreaks. Members of the interprofessional team include primary care clinicians, pathologists, infectious disease specialists, laboratory technicians, and nurses.

PCR is a nucleic acid amplification technique involving denaturation, annealing, extension, and amplification of short DNA or RNA segments. The reaction employs DNA polymerase derived from Thermus aquaticus, known as Taq polymerase. The thermostability of Taq polymerase preserves the physical and chemical integrity of nucleic acids throughout repeated high-temperature cycles, making it suitable for the PCR-based detection of bacterial and viral pathogens and for screening genetic disorders.

Laboratory technicians must be fully trained in safe sample handling to ensure test quality and prevent contamination. Personal protective equipment, including face masks, gloves, and hair caps, must be worn at all times in the laboratory. Storage and handling of solutions in pipettes, glassware, and plasticware must be performed with care to avoid exposure or contamination of DNA. Members of the interprofessional team must remain updated on current guidelines and management protocols for patients with confirmed communicable diseases.

This integrated, team-based model promotes coordinated care among all interprofessional participants, advancing outcomes for patients with infectious diseases. Patients must receive clear explanations of laboratory results and appropriate counseling regarding preventive measures and medication adherence. Education on disease transmission and personal strategies to support public health is also essential. Ongoing communication between healthcare professionals and patients fosters a therapeutic alliance that helps reduce complications, limits the spread of infection, enhances safety, and preserves quality of life.