Amplify Your Practice with Point of Care Polymerase Chain Reaction (PCR) Testing

Craig M. Lilly, MD Nathaniel Hafer PhD

Extraordinary advances in the ability to rapidly and accurately determine the coding sequences of DNA have taken place in the past 25 years. A xerox-like technology called the polymerase chain reaction, or PCR, was developed that allowed minute samples to be amplified to an amount sufficient for sequencing (decoding). This advance won its inventor, Cary Mullis, the 1993 Nobel Prize in chemistry. Subsequently, the methods for determining the sequence of code letters of DNA and RNA in clinical samples has reached a new level of convenience.

PCR technologies have led to an increasing number of clinical applications and have recently been at the forefront of public health recommendations during the COVID-19 pandemic. Here, we will briefly describe how PCR works, and its potential pitfalls, followed by a description of tests available for clinicians to diagnose and manage diseases. We will also discuss the availability of these tests in a point of care format that can provide immediate access to test results at a patisent’s home or in their physician’s office.

PCR tests detect nucleic acid sequences by cyclical amplification. The specificity of the reaction is determined by a pair of “primers” that bind to sites on opposite ends of a DNA or RNA molecule. Enzymatic addition of complementary nucleotides to those of the target sequence fill in the space between the primers and generate a complementary strand comprised of nucleotides that correspond to those of the original target — i.e. guanine corresponds to cytosine. The target and complementary strands are then separated by heating the reaction mixture and the primers and then attaching both to the original target and the newly synthesized copies to generate an exponential number of copies during subsequent amplification cycles. A key feature of the method is that the copying enzyme, called a DNA polymerase, is a special in that it can withstand the repeated heating steps. It was discovered in a primitive organism that lives in hot thermal vents at Yellowstone National Park — an example of how research based on curiosity can pay off.

The sequence of the products of PCR reactions determines their chemical properties including size, charge and molecular weight. When primer design is optimized, amplification of a single product that corresponds to a unique sequence of the target nucleic acid allows specific detection of undegraded target nucleic acid sequences in a clinical sample. Exponential amplification by increasing the number of cycles allows detection of very small numbers of undegraded target nucleic acid and is limited by inadvertent amplification of non-target sequences that compromise test specificity. Including more than one set of primers in a reaction mixture allows multiplex detection of more than one target sequence.

Traditional PCR has been supplanted by reverse transcriptase or RT-PCR in which amplification and detection are performed in a single reaction vessel. Detection of amplification- protocol- derived products, often by fluorescence or colorimetric methods, allows threshold detection for a present or absent result format or when reaction conditions are optimized such that detection signal intensity is directly related to the number of target sequences in the specimen the number of copies of the target sequence can be reported. Specificity can be increased by using a detection probe that requires sequence specific binding or the use of matrix assisted laser desorption/ionization (MALDI) technology coupled with time-of-flight mass spectrometry (TOF-MS). Many non-point of care format clinical PCR tests are classified as high complexity and require a provider order and must be performed in a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory.

The flexibility and utility of PCR has led to a large and growing number of diagnostic tests. Biorepositories, such as the UMass Center for Clinical and Translational Science’s Biospecimen, Tissue and Tumor Bank, play a key complementary role by providing clinical or nucleic acid samples from normal and diseased tissue. Accordingly, while our discussion of the clinical application of PCR is intended to provide an extensive list of PCR-based tests currently available (Table 1) from our local, state and commercial laboratories, it is not intended to provide a comprehensive list of all available tests and does not focus on gene sequencing applications dependent on PCR technologies.

Clinical applications of available PCR-based tests include measurement of resistance to antibiotics, disease-specific diagnostic testing, assessment of common pathways for drug metabolism, toxicity risk assessment, measurement of response to antiviral therapies, selection of anti-neoplastic agents, and methods for monitoring rejection of transplanted organs (Table 1).

The largest category of clinical application is diagnostic testing and the largest therapeutic area is the detection of sequences specific for infectious agents.1 The high specificity of PCR testing for infectious agents is made possible, in part, by the presence of sequences not part of the human genome and allows for more easily developed dichotomous readouts than quantitative testing of human target sequences. Limitations of test interpretation include the finding that colonizing organisms with pathogenic potential can be detected in healthy individuals by highly sensitive PCR tests. Indeed, the detection of bacterial sequences in the blood of healthy volunteers has limited the positive and negative predictive power of broad-spectrum bacterial detection PCR assays. Clinical application has been widely adopted for some tests while others have not lived up to initial reports.

The clinical utility of quantitative PCR for monitoring responses to antiviral therapies is now well established, particularly for the management of HIV therapeutics. PCR-based detection of emerging variation of HIV sequences from a patient’s blood is now standard care for guiding antiviral treatment.2 Although viral genome tests are more complex than most antibiotic susceptibility tests, their sensitivity for detecting viral mutations is of great value for understanding and tracking variants with emerging drug resistance.3 Genotypic testing also has the advantage of detecting transitional mutations that do not themselves cause drug resistance but indicate the presence of selective drug pressure that allows proactive modification of antiviral care plans.

Application of PCR monitoring to organ rejection is attractive because both the extent of inflammatory gene expression and the presence of donor-organ-specific DNA in the serum can indicate inflammation and damage of the graft, the hallmarks of rejection. Despite the advantages of these noninvasive technologies, they have not, to date, provided information equivalent to that provided by organ biopsies.4 As of May 2021, a consensus panel found noninvasive laboratory testing using gene expression profiling, or GEP; nCounter Human Organ Transplant Panel, MMDX Heart, breath testing, (a.k.a Heartsbreath); and donor-derived, cell-free DNA testing, or MyTAIHeart Tests; can aid in the diagnosis of heart transplant rejection. However, further studies will be needed to determine the utility of these molecular diagnostic assays as a replacement for routine biopsies and other aspects of long-term management of heart transplant recipients.(5)

Advances in microfluidics, detection technologies, miniaturization and the evolving availability of key reagents is enabling PCR point of care testing eliminating the need for central laboratory performance of some PCR-based tests. Experience during the COVID-19 pandemic has made the public aware of the advantages of immediate PCR test results and there is strong public support for making them available. In the absence of restrictive legislation, affordable test costs will allow accurate PCR-based tests to be performed in the office or the home. Clinical skills will need to include managing false positive and misinterpreted test results. Despite these occasional undesirable effects, we expect this wider availability of laboratory testing to lead to earlier diagnosis of many diseases and better outcomes for time-to-treatment sensitive infectious, neurological, cardiovascular and neoplastic disorders.

In summary, PCR tests are increasingly familiar in our clinical practices and daily lives and are best ordered and interpreted in the context of the clinical question that is being addressed. Patient-ordered testing represents a new starting point for provider-patient discussions. Clinical skill and an understanding of the limitations of these sensitive assays are necessary for the optimal use of PCR-based medical testing.

Craig M. Lilly, MD Professor of Medicine, Anesthesiology and Surgery, University of Massachusetts Chan Medical School, UMass Memorial Medical Center, 281 Lincoln Street, Worcester, MA 01605 email: craig.lilly@umassmed.edu

Nathaniel Hafer PhD University of Massachusetts Chan Medical School, Morningside Graduate School of Biomedical Sciences, UMass Chan Medical School Center for Clinical and Translational Science and Program in Molecular Medicine