Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts

Qinzheng Zhou; Ya Liu; Zheng Wang; Huimin Wang; Xingyao Zhang; Quan Lu

doi:10.3389/fpls.2022.856109

Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts

Front Plant Sci. 2022 Mar 18:13:856109. doi: 10.3389/fpls.2022.856109. eCollection 2022.

Authors

Qinzheng Zhou¹, Ya Liu¹, Zheng Wang¹, Huimin Wang¹, Xingyao Zhang¹, Quan Lu¹

Affiliation

¹ Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Research Institute of Forest Ecology, Environment and Nature Conservation, Chinese Academy of Forestry, Beijing, China.

Abstract

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of great significance. Traditional molecular diagnostic methods are time-consuming and require sophisticated instruments or skilled operators, which are unavailable in resource-limited settings. A specific, sensitive, and field-applicable diagnostic method is urgently needed. In this study, we developed a diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus. The false-positive signals from primer-dependent artifacts were eliminated using a probe, and base substitutions were included in the primer and probe. The entire detection process for the RPA-LFD assay can be completed under 38°C within approximately 30 min, including 15 min for crude nematode genomic DNA (gDNA) extraction and master mix preparation, 15 min for the RPA-LFD assay. This assay displayed high specificity toward B. xylophilus and showed no cross-reactions with closely related species, including Bursaphelenchus mucronatus and Bursaphelenchus doui. The sensitivity of this assay had a detection limit as low as 1 pg of B. xylophilus purified genomic DNA. Furthermore, the application of the RPA-LFD assay in simulated spiked pinewood samples showed accurate detection results. The RPA-LFD assay in this study successfully detected B. xylophilus in less than 30 min, providing a novel alternative for the simple, sensitive, and specific detection of B. xylophilus and showed potential for B. xylophilus point-of-care testing (POCT) in resource-limited areas or in field.

Keywords: Bursaphelenchus xylophilus; POCT; false positive; lateral flow dipstick; pine wilt disease; rapid diagnostic; recombinase polymerase amplification.