The cycle threshold (Ct) can be defined as the thermal cycle number needed to amplify viral RNA at which the fluorescent signal exceeds that of the background and thus passes the threshold for positivity. A typical RT-PCR assay will have a maximum of 40 thermal cycles.
Ct value and its significance
Cycle threshold (Ct) is a semi-quantitative value that tells us the approximate concentration of viral genetic material present in the patient sample following testing by RT PCR. A low Ct indicates a high concentration of viral genetic material, which is typically associated with a high risk of infectivity.
A 3-point increase in Ct value is roughly equivalent to a 10-fold decrease in the quantity of viral genetic material.
A high Ct indicates a low concentration of viral genetic material which is typically associated with a lower risk of infectivity (late in infection) but a high Ct does not rule out the possibility of a high risk of infectivity as high Ct can appear in early infection and also in inadequately collected or degraded samples.
A single Ct value in the absence of clinical context cannot be relied upon for decision-making about a person’s infectivity.
Each SARS-CoV-2 RT-PCR assays will have a slightly different limit of detection (LoD) – the lowest concentration of virus that can be reliably and consistently detected by the assay.
Working Mechanism of RT-PCR
RT-PCR detects the presence of viral genetic material in a sample but is not able to distinguish whether an infectious virus is present. The first step in SARS-CoV-2 RT PCR is to extract the viral RNA from the sample to purify, stabilize and concentrate it, to increase detection of samples containing a low quantity of virus.
The purified extract is added to a biochemical reaction mixture containing primers, nucelotide bases, enzymes and fluorescently labelled probes.
Primers attach to target regions of the viral nucleic acid, allowing the enzyme to add nucleotides to elongate a complementary DNA (cDNA) strand. The sample reaction mixture is subjected to repeated thermal cycles so that copies of the viral target are doubled per cycle leading to exponential rise. Labeled probes emit a fluorescent signal in the presence of a newly synthesized target. The earlier that exponential increase occurs, the higher the quantity of virus in the sample.
RT-PCR target gene of SARS-CoV-2
Some RT-PCRs are designed to identify a single gene target and others will detect multiple targets.
The quantity of intact virus in upper respiratory swabs will be affected by factors that are endogenous and exogenous to laboratory methods.
Laboratory exogenous factors
- The adequacy of sample collection.
- The quantity of virus at the collection site.
- The presence of inhibitors.
Laboratory endogenous factors
- The total volume of sample collection buffer/medium.
- The sample preparation method (heat, lysis methods).
- The laboratory reagent volumes used in each step of the RT-PCR process.
- The RT-PCR assay of choice.
Comparison of Ct values of different labs
As laboratories may be using different RT PCR assays, Ct values obtained in different laboratories cannot be directly compared. Ct values cannot be directly compared between assays of different types due to variation in the sensitivity (limit of detection), chemistry of reagents, gene targets, cycle parameters, analytical interpretive methods, sample preparation and extraction techniques.
References and further readings
- Introduction to testing for COVID-19. BMJ Learning
- Understanding cycle threshold (Ct) in SARS-CoV-2 RT-PCR. Public Health England