One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species.

Guangwu Lu,Zhaopeng Yang,Chuntao Zhang,Dawei Shi,Chengming Wang,Jilei Zhang,Xiaolu Li,Jens H Kuhn

doi:10.1371/journal.pone.0126281

Guangwu Lu, Zhaopeng Yang + Show 6 more

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https://doi.org/10.1371/journal.pone.0126281

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Abstract

Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct T m (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255bp, Sudan 211bp, Reston 192bp, Taï Forest 166bp, Bundibugyo 146bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

Highlights

Ebola Virus Disease (EVD), previously known as Ebola hemorrhagic fever, is an emerging infectious disease caused by a deadly virus of the family Filoviridae, genus Ebolavirus [1, 2]
Our one-step RT FRET-PCR was capable of detecting a single DNA copy and 10 RNA copies of all five Ebolavirus species (Figs 3 and 5)
The BLASTN results for each of the 8 oligonucleotides we used in the one-step RT FRET-PCR showed they were specific for the Ebolavirus species and did not cross-react with partial amplicons or other viruses in the family Filoviridae

Summary

Introduction

Ebola Virus Disease (EVD), previously known as Ebola hemorrhagic fever, is an emerging infectious disease caused by a deadly virus of the family Filoviridae, genus Ebolavirus [1, 2]. In addition to the positive controls of plasmids and transcripts described above, we prepared the VLPs containing the Ebolavirus NP RNA fragment to further verify the specificity and reverse-transcription efficiency of the established PCR in this study.

Results

Conclusion