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. 2021 Apr 1;13(4):599.
doi: 10.3390/v13040599.

Updated and Validated Pan-Coronavirus PCR Assay to Detect All Coronavirus Genera

Myndi G Holbrook  1 , Simon J Anthony  2 , Isamara Navarrete-Macias  2 , Theo Bestebroer  3 , Vincent J Munster  1 , Neeltje van Doremalen  1
Affiliations

Updated and Validated Pan-Coronavirus PCR Assay to Detect All Coronavirus Genera

Myndi G Holbrook et al. Viruses. .

Abstract

Coronavirus (CoV) spillover events from wildlife reservoirs can result in mild to severe human respiratory illness. These spillover events underlie the importance of detecting known and novel CoVs circulating in reservoir host species and determining CoV prevalence and distribution, allowing improved prediction of spillover events or where a human-reservoir interface should be closely monitored. To increase the likelihood of detecting all circulating genera and strains, we have modified primers published by Watanabe et al. in 2010 to generate a semi-nested pan-CoV PCR assay. Representatives from the four coronavirus genera (α-CoVs, β-CoVs, γ-CoVs and δ-CoVs) were tested and all of the in-house CoVs were detected using this assay. After comparing both assays, we found that the updated assay reliably detected viruses in all genera of CoVs with high sensitivity, whereas the sensitivity of the original assay was lower. Our updated PCR assay is an important tool to detect, monitor and track CoVs to enhance viral surveillance in reservoir hosts.

Keywords: PCR; coronavirus; detection; pan-CoV PCR; pandemic; reservoir host.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Primer aligned with 48 CoV sequences that represent all four genera of CoVs. Representatives for each group were chosen randomly and include all tested CoVs. (A) Watanabe and (B) Pan-CoV primers aligned with CoV representatives. Red: α-CoVs, yellow: β−CoVs, light blue: δ-CoVs, dark blue: γ-CoVs. * = in-house tested CoVs.
Figure 2
Figure 2
Limit of detection (LOD) for the Watanabe and Pan-CoV PCR assays. The LOD was determined for both assays for 15 CoVs from four genera. # = the total number of primer mismatches. * = not detected in assay.
Figure 3
Figure 3
The sum of mismatches in the primer set contribute to the sensitivity of the PCR assay. Graph displaying the sum of mismatches within primer sets against the average limit of detection (LOD) per virus. A significant correlation was found for the Watanabe primers, but not the Pan-CoV primers using a Spearman’s rank correlation coefficient. Circle = no mismatches in 3′ end primer; square = 1 mismatch in 3′ end primer; triangle = >1 mismatch in 3′ end primer.
Figure 4
Figure 4
Maximum likelihood phylogenetic tree analysis with 1000 bootstraps made in PhyML [24] displays the 430 nt product from the Pan-CoV PCR. The PCR product allows for separation into four CoV genera. * = tested CoVs in Pan-CoV PCR.
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