Polymerase Chain Reaction (PCR)

Testing Procedures

PCR procedures begin by collecting a small DNA sample in a test tube.[4] The PCR consists of 3 major phases—denaturation, hybridization or annealing, and elongation or amplification.[1] During the denaturation phase, DNA is heated to 95 °C to dissociate the hydrogen bonds between complementary base pairs of the double-stranded DNA.[1] Immediately after denaturation, the annealing process occurs; annealing involves cooling the denatured DNA at a temperature ranging from 37 to 72 °C, allowing the hydrogen bonds to reform.[4] Annealing best occurs at temperatures between 55 and 72 °C.[1]

The specific temperature is determined based on the physical and chemical properties of the specific primers used in the solution.[3] Primers are 20 to 25 nucleotides in length.[5] Annealing allows the primers to bind to the single-stranded DNA at their respective complementary sites beginning at the 3’ end of the DNA template.[1][3] Subsequently, the binding of the primers to their complementary sites on single-stranded DNA generates 2 double-stranded molecules. Finally, an optimal reaction temperature, 75 to 80 °C, best suitable for enzyme-induced DNA replication, is selected to ensure DNA polymerase activity.[3]

For DNA polymerase to function, double-stranded DNA is essential to initiate replication.[3] The enzyme then synthesizes new DNA strands in a 5′ to 3′ direction, producing strands identical to the template strands.[3] This process is repeated several times using a thermal cycler.[5] A thermal cycler is a device that controls the time and temperature of each cycle and its respective steps.[5] This process results in the amplification of multiple copies of the target DNA within the tube.[1]

After 30 to 40 cycles, repetitive cycles eventually taper off due to the limited capability of the reagent and other contributing factors such as accumulation of pyrophosphate molecules, excessive self-annealing, and the presence of PCR inhibitors in the sample.[3] Several inhibitors can affect the proper functioning of PCR. The most common PCR inhibitors are proteinase K, phenol, and EDTA.[5]

Proteinase K has the propensity to degrade Taq polymerase.[5] Other substances negatively impacting PCR tests are ionic detergents, heparin, spermidine, and hemoglobin.[5] In addition, bromophenol dyes and xylene cyanol can constitute complications in PCR testing.[5] To overcome these issues, DNA templates can be cleansed by dialysis and precipitation by ethanol. Several other strategies to clean the DNA template include using chloroform for extraction purposes and chromatography.[5]

After PCR amplification, agarose gel electrophoresis with ethidium bromide staining is typically performed to visualize the DNA.[1] The gel is then assessed in ultraviolet light.[1] An essential step in this procedure involves verifying the specificity of the results by transferring the DNA to a filter and using a probe, such as in Southern blot hybridization.[1] This step also helps eliminate nonspecific amplification, such as primer dimers.[1]

Using PCR in basic and biomedical sciences offers several advantages. Over the years, it has acquired a renowned reputation, making it the gold standard procedure for various applications.[1] PCR is known for its ability to produce rapid results in a time-efficient manner; the PCR procedure typically requires a few hours to 3 days to generate results.[1] A small sample of DNA or RNA (0.1-5 mcg) is required to undergo this reaction.[1] PCR also has the susceptibility to amplify 106 to 109 copies of DNA in a short period.[1] PCR can generate efficient amplification products following cloning and expression due to the presence of restriction sites at terminal ends.[1] 

Real-time PCR

Real-time PCR is an alternative method for analyzing small DNA segments through the shortened duration of the cycles, elimination of post-PCR procedural handling steps, implementation of fluorogenic labels, and efficient detection of emissions.[6] The discrete difference between real-time PCR and conventional PCR is the ability of real-time PCR to detect amplicons rapidly.[6] The rapid detection of amplicons in real-time PCR is accomplished through surveillance by labeling primers and fluorogenic molecules consisting of probes or amplicons.[6]

The disadvantage of real-time PCR compared to conventional PCR is that it requires opening the system to track the progression of amplicons.[6] In addition, a few fluorogenic chemicals are incompatible with the real-time PCR platforms.[6] Lastly, it is more expensive compared to conventional PCR. The aforementioned disadvantage of real-time PCR is predominantly due to the hardware incompatibilities and the accessibility of fluorogenic dyes.[6]

Reverse Transcriptase PCR 

Reverse transcription PCR (RT-PCR) is a procedure that uses messenger RNA for DNA amplification using DNA polymerase.[3] The DNA polymerase in RT-PCR is derived from retroviruses that contain RNA, generating complementary DNA. RT-PCR can be used in conjunction with conventional PCR to analyze specific gene expressions qualitatively.[3]

Real-time PCR and RT-PCR are employed simultaneously to assess the quantitative difference in gene expression among various samples.[3] During the COVID-19 pandemic, RT-PCR has been the primary diagnostic tool due to its high sensitivity, specificity, and rapidity.[7] SARS-CoV-2 samples are generally acquired from various sites in the upper respiratory tract.[7]

Samples for PCR testing may be acquired from the nasopharynx, oropharynx, nostril, and oral cavity.[7] The samples are collected through swabs, washes, and bronchoalveolar lavage.[7]

Interfering Factors

PCR has a few notable drawbacks. This test is susceptible and can detect even minor contamination in DNA or RNA, potentially leading to inaccurate results.[3] The primers designed for PCR require sequences to detect specific pathogens and genes.[3] The occasional nonspecific annealing of primers to similar, but not exact, target genes is another interfering factor.[3] The potential development of primer-dimers amplified by DNA polymerase can result in competition with PCR reagents.[3]

Results, Reporting, and Critical Findings

In PCR, amplification of DNA can be observed using fluorescent dyes that bind to double-stranded DNA or sequence-specific probes. The amplification reaction consists of a quantification cycle, Cq. Cq is the number of fractional cycles required for fluorescence to reach a threshold level for quantification.[8]

After determining Cq, a qualitative conclusion can be deduced, or a quantitative analysis may be further conducted. Cq depends on PCR efficiency; PCR efficiency involves assessing the amplification efficiency, which is explained as a fold increase per cycle with a fold value ranging from 1 to 2, with a fold value of 2 indicating 100% PCR efficiency. PCR efficiency is derived from standard and amplification curves.[8]

Standard curve PCR efficiency increases the likelihood of dilution errors, ultimately affecting the accurate quantification of several clinical and biological samples. However, individual amplification curves do not include confounding variables for the analysis of PCR efficiency, resulting in varying results compared to standard curves for the same assay. Accurate computation of the target quantity is essential for appropriate amplification efficiency, which is reflected in the analysis.[8]

A low PCR efficiency requires additional cycles to reach an appropriate quantification threshold, resulting in a higher Cq. After using a valid probe-based assay, the presence of amplification indicates that the sample contains the particular target, leading to a positive diagnosis. Due to the Poisson variation, the lack of amplification is not a valid criterion for classifying a reaction as negative.[8]

As mentioned earlier, qPCR measures DNA or RNA in various diagnostic and biological samples through the Cq. qPCR is often computed assuming all assays are 100% efficient. In addition, reporting of qPCR involves Cq, delta-Cq, or delta-delta-Cq. Efficiency correction should be used in qPCR testing to significantly and purposefully interpret biological, clinical, and diagnostic samples. Thus, it is essential to consider these factors when analyzing and reporting PCR efficiency to yield adequate results.[8]

The stage and degree of a patient's ailment can be reckoned using cycle threshold (Ct) values concurrently with clinical manifestations and disease history. Moreover, healthcare professionals can further monitor the progression of diseases and foreshadow steps to recover and resolve ailment by repeating the PCR test and generating serial Ct values. Ct values can also aid contact tracers in focusing on patients with a more elevated viral genomic load, signifying a higher risk for disease 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.[1][4][10] This technique has been frequently used to recognize various viral infectious disease microorganisms. Some of the viral pathogens detected via PCR include human papillomavirus, HIV, herpes simplex virus, SARS-CoV-2, varicella-zoster virus, enterovirus, cytomegalovirus, and hepatitis B, hepatitis C, hepatitis D, and hepatitis E.[1][3][11][7] The presence of bacterial, fungal, and parasitic organisms and various immunodeficiencies can be detected through PCR, making it an instrumental tool in clinical diagnoses and practice.[1]

The rapid identification of microbial pathogens through rapid real-time PCR allows clinicians to promptly provide tailored treatment, thus reducing hospitalizations and preventing inappropriate administration of antibiotics and, in turn, antibiotic resistance.[6] Real-time PCR has the propensity to detect specific bacterial species, such as Mycobacterium species, Leptospira genospecies, chlamydia species, Legionella pneumophila, Listeria monocytogenes, and Neisseria meningitidis.[6] Real-time PCR has also proven to effectively detect and examine antibiotic-resistant strains, such as Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, and Enterococcus.[6] Furthermore, fulminant diseases are also detected and examined early due to the high sensitivity, specificity, and rapidity of the real-time PCR test, making it the ideal procedure for medical conditions such as meningitis, sepsis, and inflammatory bowel diseases.[6]

Additional microbial pathogens infamous for causing foodborne-related illnesses, such as group B Streptococci, Mycobacterium species, Bacteroides vulgatus, and Escherichia coli, can also be identified through real-time PCR testing.[6] The rapid nature of real-time PCR allows for early detection, aiding in source tracing and helping to control current and potential outbreaks.[6] Fungal, parasitic, and protozoan pathogens, such as Aspergillus fumigatus, Aspergillus flavus, Cryptosporidium parvum, and Toxoplasma gondii, have also been identified on real-time PCR testing.[6]

PCR is also used to study the histopathology of various viral and cellular genes to comprehend and diagnose malignant human diseases.[1] In addition, PCR has been used to analyze forensic samples, point mutations, DNA sequencing, and in vitro mutagenesis.[1][5] This technique has a rapid propensity to screen and detect specific alleles, which is ideal for prenatal genetic testing for carrier status.[3] PCR also can detect the presence of disease and mutations in utero and adults.[12]

Quality Control and Lab Safety

Contamination of PCR

Due to its promising results, conventional PCR is the gold standard for screening and detecting a wide scope of scientific areas of interest. Adequate handling following PCR procedure is imperative for proper assessment of amplicon.[6] However, in conventional PCR, post-procedural improper handling can lead to the proliferation of amplicons within the laboratories.[6]

To prevent contamination of PCR, it is crucial to have a designated area of the laboratory exclusive for PCR testing to limit unnecessary turbulence within the area.[5] Face masks, gloves, and hair caps should always be worn in the laboratory to prevent contamination.[5] Preparation and storage of solutions in equipment such as pipettes, glassware, and plasticware should not be contaminated or exposed to DNA.[5]

A specific section in a freezer closest to the laminar flow hood should contain enzymes and buffers.[5] Use of any reagents should be discarded immediately.[5] A laminar flow hood with ultraviolet lights is the ideal location in a laboratory to perform PCR.[10] Equipment such as pipettes, sterile gloves, and microcentrifuge should be in the laminar flow hood.[5]

Automatic pipettes are known to cause contamination. Thus, positive displacement pipettes should be used for the proper handling of the reagents.[5] Any disposable laboratory equipment, such as pipette tips and tubes, should not be autoclaved before use.[5] In addition, disposable equipment should be used directly from its respective packaging.[5]

Before using microcentrifuge tubes consisting of reagents exclusively for PCR, centrifugation is required for approximately 10 seconds to allow the fluid to settle at the bottom of the tube, preventing contamination.[5] Post-amplification techniques should be completed at a laboratory bench instead of the designated area for PCR testing.[5]

Enhancing Healthcare Team Outcomes

Efficient use of PCR by the interprofessional healthcare team can lead to early detection of bacterial and viral pathogens, prompting earlier treatments. This approach can also further aid in preventing antibiotic resistance and viral outbreaks, respectively. The interprofessional healthcare team comprises a primary care clinician, pathologist, infectious disease specialist, lab technician, and nurses.

The PCR is a nucleic acid amplification technique that involves denaturing, renaturing, elongating, and amplifying a short segment of DNA or RNA. This process utilizes DNA polymerase derived from Thermus aquaticus, commonly known as Taq polymerase. Taq polymerase has thermostable properties that prevent the irreversible alteration of the DNA or RNA's physical and chemical properties, making it ideal for the susceptible PCR method for diagnosing a wide range of bacterial and viral infections and screening genetic diseases.

Laboratory technicians should be fully trained in safely handling and using samples to ensure quality and prevent contamination. Face masks, gloves, and hair caps should always be worn in the laboratory. Storage of solutions in their respective equipment, such as pipettes, glassware, and plasticware, should be performed with caution to prevent DNA from being exposed and contaminated. The interprofessional team should be up to date with the latest guidelines and management strategies for patients with confirmed communicable diseases.

This integrated team-based approach provides care coordination from all interprofessional team members to further advance the health of patients suffering from infectious diseases. Patients should also be thoroughly informed on laboratory findings and counseled on preventative measures and the importance of medication compliance. Patients should also be educated on disease transmission and preventive measures they can incorporate to ensure public health and safety. Continuous communication between the healthcare team and their patients can help form a therapeutic alliance to prevent complications and spread of infectious disease, ensure patient and public safety, and preserve the quality of life.