Related Experiment Video
Updated: Oct 9, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Modelling RT-qPCR cycle-threshold using digital PCR data for implementing SARS-CoV-2 viral load studies
Fabio Gentilini1, Maria Elena Turba2, Francesca Taddei3
1Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell'Emilia, Bologna, Italy.
Objectives:
To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/μL.
Methods:
A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/μL of SARS-CoV-2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy.
Results:
The model for converting the Ct values into copies/μL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance.
Conclusion:
The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity.
