Reverse-transcriptase Polymerase Chain Reaction (RT-PCR) is the main laboratory diagnostic tool used to detect active infection with SARS-CoV-2 virus. Developed in the early 1980’s, the polymerase chain reaction (PCR) test is a method of analyzing short (target) sequences of DNA by the amplification of the genetic material sufficient for identification. The sequence of genetic material in a cell to be identified is matched to a specific primer. Amplification is a process of breaking the primer-extracted DNA strand into two single strands by denaturing, usually with heat, and then building a new DNA strand by annealing each strand with a DNA polymerase enzyme. Originally created to identify DNA segments, PCR had very little utility to identify virus due to a predominance of RNA genomes. Reverse transcription PCR (RT-PCR) converts the RNA into a complimentary DNA strand (cDNA) by the enzyme reverse transcriptase. The process then follows the PCR process to amplify genetic material for identification. Conventional PCR methods have limitations when attempting to quantify pathogen load. A major development in PCR technology was the advent of real-time or quantitative RT-PCR (RT-qPCR). This technology added to the strength of PCR as a diagnostic tool as the amplification of the gene is detected in real time by the use of a fluorescent maker. The term cycle threshold (Ct) is used to identify the number of cycles of gene amplification necessary to detect the viral pathogen. Many RT-qPCR tests involve a Ct cutoff of 40 to consider the test positive for the presence of RNA. Ct values are inversely proportional to the viral load of the sample. Although many molecular diagnostic laboratories have the capabilities to conduct RT-qPCR, many only report presence or absence of disease.
The high sensitivity of detecting viral RNA by NAATs has limitations. The RT-PCR will pick up nonviable or killed virus as well as low viral load in infected individuals. The test is most effective when there is suspicion of disease either by symptoms and/or epidemiological linkage and the test is conducted close to symptom onset or contact tracing utilizing incubation periods from time of exposure. Most studies indicate that test sensitivity (ability to detect infectious disease when it is actually present) reduces with the time since onset of symptoms. Cycle thresholds over 35 may indicate that the patient is recovering or no longer infectious.
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