Point-of-care diagnostic (POCD) method for detecting Bursaphelenchus xylophilus in pinewood using recombinase polymerase amplification (RPA) with the portable optical isothermal device (POID)

PLoS One. 2020 Jan 14;15(1):e0227476. doi: 10.1371/journal.pone.0227476. eCollection 2020.

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA, Helminth / metabolism
  • Genome, Helminth
  • Limit of Detection
  • Nucleic Acid Amplification Techniques / instrumentation
  • Nucleic Acid Amplification Techniques / methods*
  • Pinus / parasitology*
  • Plant Diseases / parasitology*
  • Point-of-Care Systems
  • Recombinases / metabolism
  • Sequence Alignment
  • Tylenchida / genetics*
  • Tylenchida / isolation & purification

Substances

  • DNA, Helminth
  • Recombinases

Grants and funding

This study was supported by the ICT based researches about developing the detection method for PWD and PWN-infected pine tree (FE0702‐2017‐03) from the National Institute of Forest Science, Republic of Korea to HH. 2NCBio provided support in the form of salaries for SP. 2NCBio provided the portable optical isothermal device in development. There was no additional external funding received for this study. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.