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

Guangwu Lu; Jilei Zhang; Chuntao Zhang; Xiaolu Li; Dawei Shi; Zhaopeng Yang; Chengming Wang

doi:10.1371/journal.pone.0126281

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

PLoS One. 2015 May 27;10(5):e0126281. doi: 10.1371/journal.pone.0126281. eCollection 2015.

Authors

Guangwu Lu¹, Jilei Zhang¹, Chuntao Zhang², Xiaolu Li³, Dawei Shi², Zhaopeng Yang², Chengming Wang¹

Affiliations

¹ Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China.
² National Institute for Food and Drug Control (NIFDC), China Food and Drug Administration, Chongwen District, Beijing, 100050, P. R. China.
³ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, P. R. China.

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 Tm (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 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blood / virology
DNA Primers
Ebolavirus / genetics*
Ebolavirus / isolation & purification
Fluorescence Resonance Energy Transfer / methods
Humans
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sensitivity and Specificity

Substances

DNA Primers

Grants and funding

This project was supported by grant from the National Natural Science Foundation of China (NO: 31472225; http://www.nsfc.gov.cn/publish/portal1/) and the Priority Academic Program Development of Jiangsu Higher Education Institutions, Yangzhou, Jiangsu, P. R. China (http://jsycw.ec.js.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.