The significance of RT-PCR in COVID-19

A specific DNA sequence is detected and amplified using the enzymatic technique of polymerase chain reaction (PCR), which replicates DNA sequences. Reverse transcription PCR (RT-PCR) is a technique that combines PCR-assisted DNA amplification with reverse transcription of RNA into complementary DNA (cDNA). RNA serves as both the starting material and template for nucleic acid amplification in RT-PCR. For the diagnosis and quantification of RNA viral infections, including the current SARS-CoV-2 virus, RT-PCR is frequently utilized.

Quantitative PCR (qPCR) can be coupled with RT-PCR to assess the amount of cDNA that builds up in the reaction after each cycle of amplification. Applications for RT-transcriptional qPCR’s capability include pathogen identification, RNAi validation, gene expression analysis, and illness research. In COVID-19 diagnostic testing and research, both methods are used.

The COVID-19 diagnostic test uses RT-PCR to locate the virus in the patient’s body. The RNA genome of SARS-single-stranded CoV-2 is encoded. Therefore, the patient’s swab sample’s RNA must be completely extracted using a proper nucleic acid preparation process. The extracted RNAs are then all converted into cDNAs using the reverse transcriptase enzyme. From this point on, PCR is used to amplify and identify a specific DNA molecule, the viral cDNA of interest. The denaturation phase of the PCR process causes the initial separation of these double-stranded DNAs. The combination is then supplemented with a unique primer that can only bind to viral cDNA. Due to the primers’ specificity, only the viral cDNA and not any other cDNA in the sample will be bound by them. The separated cDNA strands are extended into two full double-stranded cDNAs in the last step by a polymerase enzyme, which is employed to add nucleotides to the ends of the primers. To amplify the DNA to the needed level for detection and further analysis, this process is cycled through several times. The patient’s swab sample is tested during this detection stage to see if the SARS-CoV-2 virus is present.

By incorporating a specific probe that will glow anytime a new DNA molecule is created, the qPCR component may be introduced to this process. With this probe, the increase in fluorescence signal can be used to follow the production of viral cDNA in real-time. We can be positive that the viral cDNA presence is much higher than the background noise when the signal reaches a predetermined threshold. The quantity of cycles needed for the signal to reach the threshold is known as the cycle threshold (Ct). Since it takes only a few cycles for the signal to surpass the threshold, a low Ct number denotes a high concentration of RNAs in the sample. Consequently, a low Ct value also indicates that the viral load in the initial sample was larger. A Ct value under 40 is regarded as a positive test for SARS-CoV-2. Numerous studies indicate that the lower the Ct number, the greater the likelihood that the patient will experience a COVID-19 condition that is more severe since there must be more viral particles in their body.

To reduce false positive and, more significantly, false negative results in COVID-19 diagnostic tests, high-specificity RT-PCR kits are required. Rapid and high throughput testing requires a method that can generate test results in a matter of hours. As a result, the need for this technology increased dramatically in 2020 as a result of the urgent requirement for COVID-19 quick testing and tracking to stop the spread. Sales of RT-PCR and RT-qPCR reagents and kits used in COVID-19 diagnostics are anticipated to increase in the near future in addition to the instruments. However, as soon as an effective vaccine against SARS-CoV-2 is made publicly accessible, this strong expansion will quickly subside. The Global Laboratory Consumables Industry 2020 study from SDi contains information on this fascinating market trend. Along with segmentation projections, vendor share, and other data, the research evaluates the global market size for both analytical and life science consumables.