Long-Range Polymerase Chain Reaction Method for Sequencing the Ebola Virus Genome From Ecological and Clinical Samples

J Infect Dis. 2018 Nov 22;218(suppl_5):S301-S304. doi: 10.1093/infdis/jiy290.

Abstract

Sequencing viral genomes during an outbreak can facilitate response and containment efforts. In this study, we describe a reverse transcription long-range polymerase chain reaction for efficient amplification and sequencing of the Ebola virus (EBOV) genome in 2 seminested reactions. We demonstrate that our method remains robust with complex biological samples by amplifying and sequencing the EBOV genome from EBOV-infected nonhuman primates (NHPs). We further demonstrate that we are able to recover viral genomes from starting concentrations as low as 103 50% tissue culture infective dose (TCID50)/mL, suggesting that this method can be employed to sequence EBOV genomes from ecologically or clinically derived samples.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Disease Outbreaks
  • Ebolavirus / genetics*
  • Genome, Viral / genetics*
  • Hemorrhagic Fever, Ebola / virology*
  • Humans
  • Macaca fascicularis / virology
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, RNA / methods