Figure 4 | Scientific Reports

Figure 4

From: Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification

Figure 4

Digital droplet PCR (ddPCR), but not bulk qRT-PCR, accurately quantifies SARS-CoV-2 viral load from patient nasopharyngeal samples without nucleic acid isolation. (a) Ct values for qRT-PCR from purified RNA (black) versus RNA prepared from crude lysate (red) shows decreased PCR efficiency without upfront nucleic acid purification of patient samples as evidenced by increase Ct values (n = 32 samples). (b) Average difference in viral load between qRT-PCR from purified RNA versus either qRT-PCR from crude lysate (red) or ddPCR from crude lysate (blue) demonstrates increased bias with qRT-PCR compared to ddPCR for low viral load (< 1000 copies/reaction, p = 0.08) but not (b′) high viral load (> 1000 copies per reaction, p = 0.94). (c) Relative quantification of viral load by qRT-PCR from purified RNA versus crude lysate demonstrates systematic underestimation of viral load. (d) Absolute quantification of viral load by ddPCR from crude lysate shows strong correlation with relative quantification by qRT-PCR from purified RNA. In (c) and (d), graphed y = x line provides a reference for perfect agreement between the two assays. All crude lysis was carried out via 1:1 dilution QuickExtract buffer followed by heating at 95 °C for 5 min as described in text.

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