Lecture 14. SARS-CoV-2 Diagnosis & Vaccination Flashcards
How can SARS-CoV-2 be diagnosed in a laboratory?
Detection of viral nucleic acid
Detection of viral antigens
Detection of viral antibodies
How can the viral nucleic acid of SARS-CoV-2 be detected?
RT-PCR (reverse transcriptase PCR)
What genes of SARS-CoV-2 could be the target for RT-PCR (as long as the correct primers are made for them)?
ORF1a/b
RdRp (RNA-dependent RNA polymerase)
S (spike protein)
E (envelope)
N (nucleocapsid)
Hel (RNA helicase)
What happens in the process of RT-PCR?
- Specimen collection
- RNA extraction/isolation
- Reverse transcription (needed because RNA virus)
- cDNA amplification
How are specimens collected for RT-PCR?
Nasopharyngeal (NP), oropharyngeal (OP) swabs
Global swab shortage during pandemic
Discomfort associated with NP collection
Trained healthcare personnel
Alternatives to NP specimens: Saliva
What is RNA highly susceptible to?
Degradation
Sample storage
Handling
What is RNA isolation and what must it follow?
Isolation of RNA is the initial step of the RT-PCR assay and critical for the assay’s reproducibility and biological relevance
Some important points to consider here is that unlike DNA, RNA is highly susceptible to degradation; sample storage, handling, and RNA isolation must follow optimised protocols to minimise degradation at each step
What happens in one-step RT-PCR?
In a one-step assay, reverse transcription and PCR amplification are consolidated into one reaction utilising a single tube and buffer for RT and PCR steps
What are the advantages of one-step RT-PCR?
Shorter time, reproducible results, this type of assay is suitable for high-throughput diagnosis, and may reduce risk of cross-contamination and human error by limiting sample management
What happens in two-step RT-PCR?
The reactions are done sequentially in separate tubes with independently optimised buffers
What are the advantages of two-step RT-PCR?
Although more time consuming is considered more sensitive and has lower detection limits
Which genes or regions of SAR-CoV-2 should be targeted by RT-PCR?
Genes of SARS-CoV-2 that are highly conserved and abundantly expressed genes should be targeted by the RT-PCR primers
What specific genes is it recommended for RT-PCR to target?
Assays should target and detect viral nucleocapsids genes N
What are the positive controls for RT-PCR?
Samples that have been previously validated as positive as positive control or you can spike a solution with synthetic SARS-CoV-2 RNA and use it as positive control]
Negative controls important too
Does the detection of Viral RNA by RT-PCR demonstrate the presence of infectious virus?
Not really
The presence of the nuclear acid doesn’t confirm the virus is present
It has been shown that patients who have recovered can be persistently PCR-positive but they are non-infectious, which is confusing for quarantine and control
