With the onset of the novel coronavirus outbreak it became clear that rapid, definitive means of detecting virus would be essential for identifying infected individuals and for supporting diagnostic and treatment efforts. As the pandemic progressed, more and more tests received FDA approval and Emergency Use Authorization, the majority of which were designed around nucleic acid detection technologies and the polymerase chain reaction (PCR) technique.
Although there were initial difficulties in testing logistics and test performance, the real-time RT-PCR approach has proven essential as a first-line defense against COVID-19 spread. Despite its importance, however, certain details of the approach have lacked adequate explanation and, in some cases, have led to misinterpretations. Understanding the strengths and limitations of real-time RT-PCR is essential for examining the current virus outbreak and for those that may emerge in the future.
What is real-time reverse transcriptase-PCR (RT-PCR)?
Nucleic acid approaches have become rapid and reliable technologies, and polymerase chain reaction (PCR) is now considered the “gold standard” for viral detection. Accordingly, real-time reverse-transcriptase-PCR (RT-PCR) tests were created and deployed for SARS-CoV-2 detection owing in part to their high sensitivity and specific combined with relatively simple quantitative power.
For review, PCR entails a cycle of reactions that can amplify as little as a single DNA fragment to millions of identical copies. The essential stages include the melting phase, in which sample template DNA is separated into single strands and prepared for amplification. The annealing phase at lower temperature allows specific primers to bond with complementary sequences on the template. The extension phase then facilitates extension of the primers through incorporate of nucleic acids complementary to template. Repeat cycling of these stages results in sequence replication, proceeding at an exponential rate until then reagents are consumed or the reaction is stopped. For a more detailed description, view this link.
Reverse transcriptase-PCR (RT-PCT) is an alternative form of PCR in which the template is initially comprised of RNA rather than DNA. So called retroviruses which include the SARS viruses, HIV, and many others contain RNA as their genetic material. Analyzing this RNA first requires the use of reverse transcriptase to formulate the complementary DNA or cDNA, prior to the PCR reaction.
Real-time PCR, also known as qPCR, is an incredibly powerful technique that can not only to detect DNA but determine its quantity in a sample. Taking advantage of the sensitivity, real-time PCR can test for the mere presence of virus in a human sample. Perhaps more importantly, the technique can quantitate the amount or viral load, which may in effect correlate to the stage and severity of viral infection as well as the potential for viral spread.
The general concept is the same as PCR -- with modifications. DNA is amplified by complementary primers and undergoes exponential phase of production, eventually reaching a plateau phase. A major difference is the PCR reaction is measured in real-time during the exponential phase, typically by measuring fluorescence produced from probes during the polymerization (extension) process. This is in contrast to tradition PCR, where fluorescence is measured at the end of the reaction process (the plateau phase) in a process called end-point detection. As a net result, real-time PCR offers true quantitative information -- not simply detection for the presence of DNA.
What are the requirements of the real-time RT-PCR COVID-19 test?
A successful real-time RT-PCR for diagnostic use requires several quality control and design features.
First off, the primers for the reaction must be complementary to stable and accessible regions of the target template DNA. Stable meaning areas not prone to viral mutation among samples and accessible meaning relatively free from extensive secondary structure or binding of the template to itself thereby creating potential obstacles for reaction efficiency. These considerations are typically addressed through empirical research, testing, or bioinformatic analysis. Primers are arguably the single most critical component of a reliable PCR assay, as their properties directly control the specificity and sensitivity.
A successful test also requires a negative control in a parallel reaction, in which no DNA template is included. This control should not produce PCR product, and if it does, the primers may not be of sufficient specificity or the reaction reagents may be contaminated. In addition, and internal control of decoy template may be necessary as a test for contamination and the occurrence of false-positives.
Finally, a test should include a positive control in which a known amount of DNA template is run in parallel with the test reaction. A series of positive controls with known quantities can be used to create a standard curve, which enables accurate calculation of test viral DNA amounts when data sets are compared.
What are the limitations of the real-time RT-PCR COVID-19 test?
In consideration of the above requirements, the success of the test requires sampling procedures which ensure accuracy and reliability of analysis. The samples should be taken from tissues or surfaces in a consistent manner. Sample preparation may require optimization and precision in nucleic acid extraction methods. The reagents and buffers must be of high quality and purity. The testing environment must be from possible contaminants such as RNAse or DNase or the presence of virus or interfering DNA.
The quantitative data is only as accurate and reliable as the reagents and controls. Primers must be optimized for specificity and reference standard DNA, for the positive control standard curves, should come from certified sources.
The real-time PCR equipment must be functional and free-from errors such that clean and reliable data production is achieved. Real-time PCR runs can take several hours depending on the reaction complexity, which can be addressed by multiplexing samples and increasing parallel throughput, provided appropriate quality control measures are taken.
Several details challenged the initial rollout and effectiveness of real-time RT-PCR tests in the COVID-19 emergency, including suboptimal primers, inappropriate reference standards, and contaminated reagents and samples.
Non-standard sampling techniques were beyond the control of the test, having more to do with the lack of effective nasal throat swabbing and knowledge regarding timing of infection and severity. Research and clinical studies have shed much light on the course of infection, the proper sampling methods and timing, and other important considerations. As detailed in a recent review on the subject, much work is left to be done.
Despite the early challenges, the development of optimized and standardized methods have given rise to comprehensive guidelines for real-time RT-PCR testing published by the CDC, a validated CDC diagnostic for use with standard equipment, and over 60 FDA-approved molecular diagnostic platforms based upon the technique.