Polymerase Chain Reaction (PCR)

Testing Procedures

Polymerase chain reaction procedures begin with the collection of a small sample of DNA in a test tube.[4] PCR consists of three major phases: denaturation, hybridization/annealing, elongation/amplification.[1] During the denaturation phase, DNA is heated to 95 celsius (C) to dissociate the hydrogen bonds between complementary base pairs of the double-stranded DNA.[1] Immediately following denaturation, the process of annealing occurs; annealing involves cooling the denatured DNA at a temperature ranging from 37-72 C allowing for the hydrogen bonds to reform.[4] Annealing best occurs at temperatures between 55 C to 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 20-25 nucleotides in length.[5] Annealing allows for 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 two double-stranded molecules. Finally, an optimal reaction temperature, 75-80 C, that is best suitable for enzyme-induced DNA replication is selected to ensure DNA polymerase activity.[3]

In order to initiate the functionality of DNA polymerase, double-stranded DNA is mandatory for the occurrence of replication.[3] Thereafter, DNA polymerase synthesizes DNA in a 3’ to 5’ direction producing strands identical to the template strands.[3] This procedure is repeated several times via 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 ultimately leads to several duplicated DNA available in the tube.[1]

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

Proteinase K has the propensity to break down Taq polymerase.[5] Other substances that can negatively impact PCR tests are ionic detergents, heparin, spermidine, and hemoglobin.[5] Additionally, 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]

Following the aforementioned steps of PCR, the next step includes agarose gel electrophoresis using ethidium bromide.[1] Subsequently, the gel is assessed in ultraviolet light.[1] An imperative step in this component of the procedure requires examining the specificity of the results via transferring to a filter and implementation of a probe such as southern blot for hybridization.[1] Lastly, it removes the amplification of primer dimers.[1]

There are various advantages of using PCR in basic and biomedical sciences. Over the years, it has acquired a renowned reputation making it the gold standard procedure for a multitude of reasons.[1] Firstly, it 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 necessitated to undergo this reaction.[1] PCR also has the susceptibility to amplify  106 to 109 copies of DNA in a short period of time.[1] PCR has the ability to 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 to examine small segments of DNA  via the shortened duration of the cycles, elimination of post-PCR procedural handling steps, implementing fluorogenic labels as well as efficient detection of emissions.[6] The discrete difference between real-time PCR and conventional PCR is the ability of real-time PCR to rapidly detect amplicons.[6] The rapid detection of amplicons in real-time PCR is accomplished via surveillance by labeling primers, and fluorogenic molecules consisting of probes or amplicons.[6]

The disadvantage of real-time PCR in comparison to conventional PCR is that it requires opening the system to track the progression of amplicons.[6] In addition, there are a few fluorogenic chemicals that are not compatible with the real-time PCR platforms.[6] Lastly, it is more costly than 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 transcriptase-polymerase chain reaction (RT-PCR) is a procedure that uses mRNA for DNA amplification via DNA polymerase.[3] The DNA polymerase in RT-PCR is expressed by retroviruses that consist of RNA eliciting complementary (cDNA). Conventional PCR and RT-PCR can be conjunctively used to study specific gene expressions from a qualitative standpoint.[3]

To date, 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 main 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 via swabs, washes, and bronchoalveolar lavage.[7]

Interfering Factors

There are a few drawbacks of polymerase chain reaction. This test is highly sensitive and has the susceptibility to detect the slightest contamination in DNA and RNA yielding inaccurate results.[3] The primers designed for PCR require sequence to detect specific pathogens and genes.[3] The occasional occurrence of non-specific annealing of primers to similar, but not exact, target genes is another interfering factor.[3] The potential development of primer-dimers (PD) 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 with fluorescent dyes that bind to double-stranded DNA or probes that are sequence-specific. The amplification reaction consists of a quantification cycle, Cq. Cq is described as the number of fractional cycles necessitated for the fluorescence to meet the preset for quantification.[8]

After determining Cq, a qualitative conclusion can be deduced, or a quantitative analysis may be further conducted. Cq is dependent on PCR efficiency; PCR efficiency involves assessment of the amplification efficiency which is explained as fold increase/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 curves 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 that will be reflected in the analysis.[8]

A low PCR efficiency requires additional cycles to reach an appropriate quantification threshold resulting in a higher Cq. The presence of amplification following the utilization of a valid probe-based assay is indicative of the sample containing the particular target and subsequently concluding it as diagnostically positive. Due to the Poisson variation, the lack of amplification is not a valid criterion to classify a reaction as negative.[8]

As mentioned earlier, qPCR measures DNA or RNA in various diagnostic and biological samples via the Cq. qPCR is often computed with the assumption that all assays are 100% efficient. Additionally, reporting of qPCR involves Cq, delta-Cq, or delta-delta-Cq. For significant and purposeful interpretation of biological, clinical, and diagnostic samples, efficiency corrected should be utilized in qPCR testing. Thus, it is essential to consider these factors when interpreting and reporting PCR efficiency to yield adequate results.[8]

The stage and degree of a patient's ailment can be reckoned with by utilizing cycle threshold (Ct) values concurrently with clinical manifestations and disease history. Moreover, healthcare professionals can further surveillance 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 to focus on patients with a more elevated viral genomic load, signifying a higher risk for disease transmission.[9]

Clinical Significance

PCR is used in basic and biomedical sciences which has substantial laboratory and clinical significance due to its high sensitivity, specificity, and rapidity.[1][4][10] It has been frequently used to recognize various viral infectious disease microorganisms. Some of the viral pathogens detected via PCR include human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), SARS-CoV-2, varicella-zoster virus (VZV), enterovirus, cytomegalovirus (CMV), and hepatitis B (Hep-B), hepatitis C (Hep-C), hepatitis D (Hep-D), and hepatitis E (Hep-E). [1][3][11][7] The presence of bacterial, fungal, parasitic organisms and various immunodeficiencies can be detected via PCR making it an instrumental tool in clinical diagnoses and practice.[1]

Rapid detection of microbial pathogens via rapid real-time PCR allows for clinicians to promptly administer tailored treatment thus, reducing hospitalizations, preventing inappropriate administration of antibiotics, 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 meningitides.[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 (IBD).[6]

Additional microbial pathogens that are infamous for causing food-borne related illnesses such as group B streptococci, mycobacterium species, Bacteroides vulgatus, and escherichia coli (e.coli) can also be identified via real-time PCR testing.[6] Due to the rapid nature of real time-PCR tests, earlier detection can aid in tracing its source, in turn, controlling existing and potential outbreaks.[6] Fungal, parasitic, and protozoan pathogens have also been identified on real-time PCR testing such as aspergillus fumigatus and aspergillus flavus, cryptosporidium parvum, and toxoplasma gondii. [6]

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

Quality Control and Lab Safety

Contamination of PCR

Conventional PCR is the gold standard for screening and detecting a wide scope of scientific areas of interest due to its promising results. 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 amplicon 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 be worn at all times in the laboratory to prevent the occurrence of contamination.[5] Preparation and storage of solutions in equipment such as pipettes, glassware, and plasticware should not be contaminated or exposed with DNA.[5]

A specific section in a freezer, that is 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 (UV) lights is the ideal location in a laboratory to perform PCR.[10] Equipment such as pipettes, sterile gloves, and microcentrifuge should be present within the laminar flow hood.[5]

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

Prior to using microcentrifuge tubes consisting of reagents exclusively for PCR, centrifugation is necessitated for approximately 10 seconds to allow the fluid to settle at the bottom of the tube preventing the occurrence of contamination.[5] Post amplification techniques should be completed at a laboratory bench as opposed to 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 can also further aid in preventing antibiotic resistance and viral outbreaks, respectively. The interprofessional health care team comprises a primary care physician, pathologist, infectious disease specialist, lab technician, and nurses.

The polymerase chain reaction is a nucleic acid amplification testing procedure that consists of denaturing, renaturing, elongating, and amplifying a short segment of DNA or RNA. This is implemented by incorporating DNA I polymerase, which is derived from Thermus aquaticus, also known as Taq polymerase. Taq polymerase consists of thermostable properties preventing the irreversible alteration of the DNA or RNA physical and chemical properties, making it ideal for the highly sensitive polymerase chain reaction procedure for diagnosing a wide range of bacterial and viral infections, as well as screening genetic diseases.

Laboratory technicians should be fully trained in the safe handling and use of samples to ensure quality and prevent contamination. Face masks, gloves, and hair caps should be worn at all times in the laboratory. Storage of solutions in their respective equipment (pipettes, glassware, plasticware) should be done with caution to prevent DNA from being exposed and contaminated. The interprofessional team of healthcare providers 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 is needed. 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 communicable disease, ensure patient and public safety, and preserve the quality of life.