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Background
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) otherwise known as 2019-nCov, or COVID-19 is a positive-sense single-stranded RNA virus.
While there may be other ways to test for presence of the virus in patients (Immunoassays and CT scans), Polymerase Chain Reaction (PCR) based assays are currently the fastest and most accurate way to achieve testing at scale for current patients who have presence of active virus. PCR-based techniques for COVID-19 testing is currently the focus of this site.
General Workflow
General Workflow
SAMPLE COLLECTED FROM PATIENTS: Sample collected through swabbing of the nose, throat, or collected from sputum or brocheoalveolar lavage.
NUCLEIC ACID EXTRACTION: Nucleic acid is purified from a patient sample. This includes single stranded RNA (ssRNA) from the virus.
REVERSE TRANSCRIPTION: This is converted into double-standard DNA (dsDNA), also known as complimentary DNA (cDNA), using the enzyme reverse transcriptase.
PCR: This DNA is then detected using PCR (real-time PCR or standard PCR); using DNA primers specific to COVID-19 strains. This is either done by RT-PCR or PCR:
For real-time (quantitative) PCR (RT-PCR), fluorescent DNA probes within the COVID19 cDNA are used to quantify the amount of viral cDNA. If amplification of the COVID19 DNA occurs through the PCR reaction, a fluorescent signal is produced which is detected by the RT-PCR instrument, leading to quantification of the cDNA, and hence, detection of the virus.
For PCR detection, following the reverse-transcriptase reaction, the sample is followed by standard PCR. The Amplified PCR product is then run on a standard agarose gel for detection.
Considerations
Considerations
While in principal, the above RT-PCR test is relatively straight-forward from a molecular biology point-of-view, there are many things to address that affect testing quality.
Objective of the test - are you looking to see if a person has recently contracted and has active virus, or whether a person has previously been exposed?
Experimental design (e.g. accounting of positive and negative controls)
Quality of patient sample (correct procedure of sample acquisition, sufficient material, contamination, etc.)
Quality components of testing material (reagents, containers, enzymatic grade, chemical purity).
Training/experience of the staff conducting tests, along the complete workflow.
Validation of test protocol and reagents (comparison with known standard tests)
It must be noted, that given the urgency of deployment of COVID-19 testing, many test kits and protocols have been used, and even approved by some regulators, without complete validation. In an ideal world, test would have been through rigorous testing to ensure quality, reproducibility, high sensitivity, high specificity, etc. to ensure a reliable test result that can be trusted.
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