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. 2020 Sep;46(3):957-964.
doi: 10.3892/ijmm.2020.4673. Epub 2020 Jul 13.

Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection

Luca Falzone  1 , Nicolò Musso  2 , Giuseppe Gattuso  3 , Dafne Bongiorno  2 , Concetta Ilenia Palermo  4 , Guido Scalia  2 , Massimo Libra  3 , Stefania Stefani  2
Affiliations

Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection

Luca Falzone et al. Int J Mol Med. 2020 Sep.

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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Figures

Figure 1
Figure 1
Gene structure of SARS-CoV-2. The position of the main primers and probes proposed for the identification of the virus are shown. Pink lines are related to WHO-approved probes; blue lines are related to CDC-approved probes (8). WHO, World Health Organization; CDC, Center for Disease Control and Prevention.
Figure 2
Figure 2
Schematic workflow of RT-qPCR and ddPCR experiments. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; ddPCR, droplet digital PCR.
Figure 3
Figure 3
Linear regression analysis of β-actin ddPCR and RT-qPCR data. (A) Linear regression of β-actin values considering all dilutions of sample 1; (B) Linear regression analysis of β-actin values without the inhibited undiluted sample 1. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; ddPCR, droplet digital PCR.
Figure 4
Figure 4
(A) EvaGreen ddPCR absolute quantification of SARS-CoV-2 N gene in rhino-pharyngeal swabs; (B) Probe ddPCR absolute quantification of SARS-CoV-2 N gene in rhino-pharyngeal swabs; (C) Amplitude signal of SARS-CoV-2 N gene positive droplets obtained with Probe ddPCR. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; ddPCR, droplet digital PCR.
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