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. 2023 Apr 17;15(4):987.
doi: 10.3390/v15040987.

Development of a Pan- Filoviridae SYBR Green qPCR Assay for Biosurveillance Studies in Bats

Jessica Coertse  1   2 , Marinda Mortlock  2 , Antoinette Grobbelaar  1 , Naazneen Moolla  1   2 , Wanda Markotter  2 , Jacqueline Weyer  1   2   3
Affiliations

Development of a Pan- Filoviridae SYBR Green qPCR Assay for Biosurveillance Studies in Bats

Jessica Coertse et al. Viruses. .

Abstract

Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3-31.7 copies/reaction and was evaluated against the field collected samples. The assay's performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.

Keywords: SYBR Green; bats; pan-filovirus; qPCR; surveillance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Graphic representation of the synthetic constructs designed for use as assay controls. Each construct consists of a 537-nucleotide partial nucleoprotein sequence of the selected viral species (Table 1). The expected amplicon size for the filovirus products was 157 bp. The control tag sequence was situated at position 34–51 within the assay amplification region. The SP6 promotor was located at the end of the synthetic construct to allow for the synthesis of RNA transcripts.
Figure 2
Figure 2
Filovirus SYBR Green qPCR data analysis workflow. The diagram depicts the workflow to assess individual samples at several steps to determine the additional analyses required for suspected positive samples. Results indicated in light blue represent negative samples and no further analyses were needed. Findings indicated in light green require further data analyses indicated with arrows. *: Numerical values of the Cq/Tm provided; #: Visual representation of the amplification/melt curve plots; $: Analyses of nucleotide sequences using the BLAST function of the National Center for Biotechnology Information (NCBI) database (accessible online at https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 January 2023)).
Figure 3
Figure 3
Average melting temperatures of the amplified filovirus standard templates (100–109 copies/reaction). Error bars indicate the standard deviation.
Figure 4
Figure 4
Standard curves for the serially diluted (105–109 copies/reaction) standard templates.
Figure 5
Figure 5
Comparison of the Cq values for the probe-based assay and the average Cq values for the SYBR Green assay for the titrated (TCID50/mL) serially diluted Ebola virus.
Figure 6
Figure 6
Comparison of the Cq values for the probe-based assay and the average Cq values for the SYBR Green assay for the titrated (TCID50/mL) serially diluted Marburg virus.
Figure 7
Figure 7
Comparison of the Cq values for the probe-based assay and the average Cq values for the SYBR Green assay for the negative bat sera spiked with the titrated (TCID50/mL) serially diluted Ebola virus.
Figure 8
Figure 8
Comparison of the Cq values for the probe-based assay and the average Cq values for the SYBR Green assay for negative bat sera spiked with the titrated (TCID50/mL) serially diluted Margburg virus.
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References

    1. Kuhn J.H., Adkins S., Agwanda B.R., Al Kubrusli R., Alkhovsky S.V., Amarasinghe G.K., Avšič-Županc T., Ayllón M.A., Bahl J., Balkema-Buschmann A., et al. 2021 Taxonomic Update of Phylum Negarnaviricota (Riboviria: Orthornavirae), Including the Large Orders Bunyavirales and Mononegavirales. Arch. Virol. 2021;166:3513–3566. doi: 10.1007/s00705-021-05143-6. - DOI - PMC - PubMed
    1. Shi M., Lin X.-D., Chen X., Tian J.-H., Chen L.-J., Li K., Wang W., Eden J.-S., Shen J.-J., Liu L., et al. Author Correction: The Evolutionary History of Vertebrate RNA Viruses. Nature. 2018;561:E6. doi: 10.1038/s41586-018-0310-0. - DOI - PubMed
    1. Emanuel J., Marzi A., Feldmann H. Advances in Virus Research. Volume 100. Elsevier Inc.; Amsterdam, The Netherlands: 2018. Filoviruses: Ecology, Molecular Biology, and Evolution. - PMC - PubMed
    1. Siegert R., Shu H.-L., Slenczka W., Peters D., Müller G. Zur Ätiologie Einer Unbekannten, von Affen Ausgegangenen Menschlichen Infektionskrankheit. DMW—Dtsch. Med. Wochenschr. 1967;92:2341–2343. doi: 10.1055/s-0028-1106144. - DOI - PubMed
    1. Languon S., Quaye O. Filovirus Disease Outbreaks: A Chronological Overview. Virol. Res. Treat. 2019;10:1178122X1984992. doi: 10.1177/1178122X19849927. - DOI - PMC - PubMed

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