Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus

Thomas R. Shelite, Nathen E. Bopp, Abelardo Moncayo, Erin S. Reynolds, Saravanan Thangamani, Peter C. Melby, Karen Bloch, Patricia V. Aguilar, Bruno L. Travi

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/μL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Original languageEnglish (US)
Pages (from-to)110-115
Number of pages6
JournalVector-Borne and Zoonotic Diseases
Issue number2
StatePublished - Feb 2021


  • clinical samples
  • heartland virus
  • isothermal recombinase polymerase amplification
  • lateral flow assay
  • point-of-care assay

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases
  • Virology


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