PCR as a diagnostic tool for COVID-19
PCR stands for Polymerase Chain Reaction. Under the current global pandemic situation, PCR has become one of the most effective methods of controlling the pandemic in means of accurate and early detection of COVID-19. Simply, a selected region of a DNA molecule is identified and amplified in PCR. The target DNA is known as the template. Two primers are used in the process which are short oligonucleotide sequences that can hybridize with the template. Those two primers are the forward primer and the reverse primer that flank the target region. To amplify the desired region from the template, DNA polymerase enzyme, PCR buffer, deoxyribonucleotides (dNTPs), and MgCl2 are needed in addition to primers. PCR is completed by the repetition of the thermal cycles which contains three steps: DNA denaturation, primer annealing, and DNA synthesis.
Diagnosis of COVID-19 involves a special type of PCR. It is the quantitative reverse transcription PCR (RT-qPCR). In conventional PCR, the result is determined at the end of the process, but in RT-qPCR the result is analyzed while the PCR reaction proceeds. One important feature here is the use of reverse transcriptase enzyme in addition to other conventional PCR constituents.
The causative agent of COVID-19 is a virus called Severe Acute Respiratory Syndrome — related Coronavirus 2 (SARS-CoV-2) which belongs to the family Coronaviridae. Positive sense single stranded RNA acts as the genome. Therefore, RNA should be detected from the samples for the diagnosis of COVID-19. These samples are collected from the upper and lower respiratory tracts, such as nasopharyngeal and oropharyngeal swabs, bronchoalveolar lavage, and tracheal aspirates. From these, the nasopharyngeal swabs are of high importance. Then viral RNA can be extracted from the sample using approved viral isolation kits, and purified before conducting the PCR. As mentioned before, since PCR amplifies DNA molecules, the RNA present in SARS-CoV-2 samples should be converted to DNA. For that purpose, a reverse transcriptase enzyme is used in RT-qPCR. It catalyzes the reverse transcription process where RNA molecules are converted to DNA molecules. The resulting complementary DNA strand (cDNA) acts as the template for DNA polymerase enzyme to amplify DNA using primers. These primers can target a specific section of the viral genome including the envelope gene, the RNA dependent RNA polymerase (RdRP) gene, and the nucleocapsid gene. To date, various primer and probe sets are used for the detection in several countries. Some laboratories use multiplex PCR which targets and detects different regions of the SARS-CoV-2 genome simultaneously.
RT-qPCR is widely used due to its many advantages. It has high sensitivity, specificity and allows early and rapid detection of very low viral loads. It is a cost-effective and an efficient method as compared to the expensive antibody based ELISA assays. But sometimes these can lead to false-positive results by cross-reactivity of primers with contaminants and other co-infecting viruses or bacteria. RNA viruses have high mutation rates that rapidly lead to variants with changes in the genome. These variants cannot be identified using existing primer-probe sets which may also lead to false-positive results. Apart from these, the variation in viral load, timing, sample collection, and handling failures may also cause false-positive results. There is a potential for exposure to disease and biological safety hazards during transport and sample handling. Other drawbacks are that it is a labor intensive and time consuming process. This may be addressed with automated PCR — a high throughput, and highly reproducible method to save time and effort.
1. D’Cruz, Roshan J., Arthur W. Currier, and Valerie B. Sampson. 2020. “Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2).” Frontiers in Cell and Developmental Biology 8(June): 1–11.
2. Ekrami, Elena et al. 2020. “Potential Diagnostic Systems for Coronavirus Detection: A Critical Review.” Biological Procedures Online 22(1): 1–18.
3. Jayamohan, Harikrishnan et al. 2021. “SARS-CoV-2 Pandemic: A Review of Molecular Diagnostic Tools Including Sample Collection and Commercial Response with Associated Advantages and Limitations.” Analytical and Bioanalytical Chemistry 413(1): 49–71.
4. Kumar, Ramesh, Suman Nagpal, Samander Kaushik, and Sanjay Mendiratta. 2020. “COVID 19 Diagnostic Approaches: Different Roads to the Same Destination.” VirusDisease 31(2): 97–105. https://doi.org/10.1007/s13337-020-00599-7.