TY - JOUR
T1 - Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection
AU - Falzone, Luca
AU - Musso, Nicolo
AU - Gattuso, Giuseppe
AU - Bongiorno, Dafne
AU - Palermo, Concetta Ilenia
AU - Scalia, Guido
AU - Libra, Massimo
AU - Stefani, Stefania
PY - 2020
Y1 - 2020
N2 - 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-2N 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.
AB - 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-2N 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.
KW - COVID-19
KW - ddPCR
KW - Diagnosis
KW - RT-qPCR
KW - SARS-CoV-2
KW - Sensitivity
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U2 - 10.3892/ijmm.2020.4673
DO - 10.3892/ijmm.2020.4673
M3 - Article
C2 - 32705153
AN - SCOPUS:85088515588
VL - 46
SP - 957
EP - 964
JO - International Journal of Molecular Medicine
JF - International Journal of Molecular Medicine
SN - 1107-3756
IS - 3
ER -