The Importance of Sensitivity in SARS-CoV-2 Real-Time RT-PCR Assays

Accurate detection of SARS-CoV-2 is critical for both patient treatments, as well as control of the COVID-19 pandemic. One important factor to consider when deciding upon a real-time reverse transcription polymerase chain reaction (RT-PCR) assay to use for detection of SARS-CoV-2 is the Limit of Detection (LOD).

Viral loads vary as much as a billion-fold between patient samples, with the highest viral load per patient occurring 5-6 days after infection.  Patients having higher viral loads are known to shed more whole virus making them more contagious, and more apt to spread disease.  The risk of getting infected with any virus is based on the equation: Successful Infection = Exposure to Virus x Time.  Experts estimate that as little as 1,000 copies of SARS-CoV-2 are needed to cause an infection.

The LOD of a real-time RT-PCR assay is the minimum amount of target that can be detected with a stated probability. In molecular diagnostics we commonly report results and make conclusions at a confidence of 95%. The LOD is the cut-off value over which the sample can be considered positive.

Why is the LOD of a SARS-CoV-2 Real-Time RT-PCR Assay Important?

Viral load is a measure of the number of viral particles present in an individual. Previous studies demonstrated that viral loads in throat swab and sputum samples peak at around 5–6 days after symptom onset. Samples taken from an individual with a recently acquired SARS-CoV-2 infection can have low viral loads. If the viral load of a sample is lower than the LOD or cut-off value of a real-time RT-PCR assay, a false negative result will be obtained. Assays with a low LOD and lower cut-off values are more likely to detect SARS-CoV-2 in samples with low viral loads which may be missed by assays with higher LODs.

Comparing LODs Between Real-time RT-PCR Assays

Unfortunately comparing the LODs of various real-time RT-PCR assays authorized under FDA EUA is not as easy as it sounds because companies report their assay LOD using different measurements. A lot of the existing real-time RT-PCR assays authorized under the FDA EUA for the detection of SARS-CoV-2 describe their LOD in copies/mL in the sample. However, some companies report LOD in genome equivalents/mL. A genome equivalent is the amount of DNA necessary to be present in a purified sample to guarantee that all genes will be present. This number increases with the total genome size of an organism and can be calculated by converting the size of a genome in base pairs to micrograms of DNA.


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Jones, T. C., et al. (2020) An analysis of SARS-CoV-2 viral load by patient age. Retrieved May 28, 2020, from

Pan, Y., Zhang, D., Yang, P., Poon, L. L. M., & Wang, Q. (2020). Viral load of SARS-CoV-2 in clinical samples. The Lancet Infectious Diseases, 20(4), 411–412. doi: 10.1016/s1473-3099(20)30113-4

To, K. et al., 2020. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. The Lancet Infectious Diseases, 20(5), pp.565-574.

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