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Method Overview:
Method Overview:
As mentioned earlier, RT-PCR is currently the Gold Standard diagnostic method used for COVID-19 diagnostics. This page delves into greater detail in regards to the process of RT-PCR, the specific primers/probes used along with the genes specific to the coronavirus, in addition to how to read an RT-PCR assay. For more background information on RT-PCR visit the Standard Diagnostic Methods page.
Steps of RT-PCR
Steps of RT-PCR
Figure 1: This video provides an easy understanding and visual animation of the steps for RT-PCR for the COVID-19 virus. Steps include: RNA extraction and purification (1:20), real time RT-PCR (mastermix: RT, dNTPs, forward primer, reverse primer, Taqman probe, DNA polymerase) (3:30), SARS-CoV-2 genome with the primers and probes (4:29), Reverse Transcription (4:38), PCR (Denaturation, Extension, Annealing) (5:15), Fluoresence detection (5:30), Measurement of fluorescent signal (7:31). Disclaimer: the video voiceover is a robot, therefore some words are mispronounced. Additionally, here is the link to this video: https://www.youtube.com/watch?v=-fc5VXTHkN8
Visual of SARS-CoV-2 Genome
Visual of SARS-CoV-2 Genome
Figure 2: This figure represents the SARS-CoV-2 viral RNA genome. Regions on the genome that the RT-PCR assays have been designed to target are indicated by a star. Most RT-PCR assays target for the E, N or RdRP genes, whereas this image presents another gene that is specific to the SARS-CoV-2 virus which is known as the ORF8 gene. E= gene that codes for envelope protein, N= gene that codes for nucleocapsid protein, RdRP= RNA-dependent RNA polymerase. Reminder that the viral RNA is inside the viral capsid and needs to be extracted before you can do RT-PCR. Table 2 in the Primer Deisgn For Quantitative Real-Time PCR for the Emerging Coronavirus SARS-CoV-2 shows the SARS-CoV-2 genome where Table 2 provides an overview of RT-PCR assays that have been designed.
PCR Primer
PCR Primer
Primers are short, single stranded DNA sequences. During PCR, forward and reverse primers bind to the target sequence, in this case the SARS-CoV-2 genomic material, and amplify this sequence.
Probe
Probe
A probe is single stranded DNA or RNA sequence that is labeled with a chemical tag in order to track a specific sample genome. A fluorescent probe is used to chemically ID the SARS-CoV-2 genomic material.
Cycle Threshold
Cycle Threshold
The cycle threshold (Ct) is the number of cycles RT-PCR must go through for the florescent signal to reach the threshold. Ct value can be related to the relative amount of virus in a sample. Essentially tests look to see if it's positive (above threshold) or negative (below threshold).
Relevant Papers
Relevant Papers
This paper gives a detailed overview of how RT-PCR is used as a COVID-19 diagnostic method. It goes into detail about the RT-PCR protocol and what materials are required for it. This paper also discusses how samples are collected (nasal or oral swabs) and the limitations of this method. It also addresses the patient inclusion/exclusion criteria when testing RT-PCR.
This paper reviews the importance of specific primers and probes, in addition to evaluating how they are designed methodically and rationally. Moreover, it discusses how the sensitivity and specificity of the detetection process can be further improved to develop more accurate results. Ultimately, this article is significant for future research in regards to accurate diagnosis and preparedness for a potential future coronavirus.
References
References
2020. , EMERGENCY USE AUTHORIZATION (EUA) SUMMARY COVID-19 RT-PCR TEST (LABORATORY CORPORATION OF AMERICA).
CDC's Diagnostic Test for COVID-19 Only and Supplies [WWW Document], n.d. [WWW Document]. URL https://www.cdc.gov/coronavirus/2019-ncov/lab/virus-requests.html (accessed 10.20.20).
Kakhki, R.K., Kakhki, M.K., Neshani, A., 2020. COVID-19 target: A specific target for novel coronavirus detection [WWW Document]. Gene reports. URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261075/ (accessed 10.20.20).
Lan L, Xu D, Ye G, et al. Positive RT-PCR Test Results in Patients Recovered From COVID-19. JAMA. 2020;323(15):1502–1503. doi:10.1001/jama.2020.2783
Li, D., Zhang, J., Li, J., 2020. Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2 [WWW Document]. Theranostics. URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330846/ (accessed 10.20.20).
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