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. 2021 Sep:295:114185.
doi: 10.1016/j.jviromet.2021.114185. Epub 2021 May 26.

Digital PCR assay for the effective detection of COVID-19 patients with SARS-CoV-2 low viral load

Yong Sun  1 , Chengchao Ding  2 , Qingqing Chen  1 , Jiajia Xie  2 , Junling Yu  1 , Yonglin Shi  1 , Chengcheng Jiang  2 , Zhuhui Zhang  1 , Hongliang He  2 , Yinglu Ge  1 , Wenting Li  2 , Jun He  3 , Yong Gao  4
Affiliations

Digital PCR assay for the effective detection of COVID-19 patients with SARS-CoV-2 low viral load

Yong Sun et al. J Virol Methods. 2021 Sep.

Abstract

Objective: Viral nucleic acid detection by real-time reverse transcription polymerase chain reaction (qPCR) is the current standard method for diagnosis of SARS-CoV-2 infection. However, due to low viral load in some COVID-19 patients, false negative results from this method have been repeatedly reported.

Method: In this study, we compared the sensitivity and specificity of digital PCR (dPCR) in simulated samples and clinical samples with qPCR assay through a series of vigorous tests.

Results: The results showed that dPCR was more sensitive than qPCR especially for samples with low viral load (≤3 copies). In addition, dPCR had similar specificity as qPCR and could effectively distinguish other human coronaviruses and influenza virus from SARS-CoV-2. More importantly, dPCR was more sensitive than qPCR in detecting the virus in the "negative" samples from recurrent COVID-19 patients.

Conclusions: In summary, dPCR could serve as a powerful complement to the current qPCR method for SARS-CoV-2 detection, especially for the samples with extremely low viral load, such as recurrent COVID-19 patients.

Keywords: Digital PCR (dPCR); Recurrent COVID-19 patient; SARS-CoV-2; Sensitivity; Specificity.

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Conflict of interest statement

The authors have no conflicts of interests to declare.

Figures

Fig. 1
Fig. 1
Comparison of dPCR assay with qPCR assay on low and moderate viral load of simulated SARS-CoV-2 samples. (A) The positive rate of low (20 samples) and moderate (10 samples) viral load samples detected by dPCR and qPCR assays. (B) The FAM values of dPCR assay targeting N gene of SARS-CoV-2 of low and moderate viral load samples.
Fig. 2
Fig. 2
Comparison of sensitivity between qPCR and dPCR assays in detecting simulated SARS-CoV-2 samples. (A) Positive rate at each concentration of 6.0, 3.0, 1.5, 0.75, 0.375 copies per reaction. (B) dPCR detection of serial dilution of authentic SARS-CoV-2 spiked into negative matrix. Expected copies of viral RNA were plotted on the X axis versus measured FAM values on the Y axis from dPCR assay targeting N gene of SRAS-CoV-2. Data are representative of 5 independent experiments with 3 replicates for each concentration of 6, 3, 1.5, 0.75, and 0.375 copies of viral RNA. (C) Further confirmation of positive rates detected by qPCR and dPCR assays on 20 replicates of low viral load (3 copies) simulated samples.
Fig. 3
Fig. 3
Comparison of specificity between qPCR and dPCR assays. (A) The positive rate of the negative and positive samples detected by dPCR and qPCR. (B) The FAM values of the negative and low positive samples detected by dPCR.
Fig. 4
Fig. 4
Evaluation of linearity using clinical samples by dPCR and qPCR assay. The linearity of CT value of qPCR, FAM positive of dPCR with the serial dilution point ranges from 10−1 to 10−6 for the #262, #265, #341 and #344 COVID-19 patient samples.
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