Abstract
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of COVID-19 infection. Due to pre-analytical and technical limitations, samples with low viral load are often misdiagnosed as false-negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT-qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID-19 were analyzed by droplet digital PCR (ddPCR) and RT-qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)-approved probe for the SARS-CoV-2 N gene were used. SYBR-Green RT-qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT-qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT-qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10-fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT-qPCR for the diagnosis of COVID-19 infection in false-negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID-19 and the follow-up of positive patients until complete remission.
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