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

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.

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA–CRISPR/Cas12a Assay

“…To this end, we diluted our in vitro-transcribed TMV RNA down to femtomolar concentrations and subjected the serial dilutions to our iSCAN-OP detection assay with the S3 set of RPA primers together with a ssDNA FAM fluorescent reporter to measure fluorescence in real-time. Our results indicate that our CRISPR-based detection assay can detect viral RNA down to the picomolar range, however, longer reaction time can improve the detection limit of the assay down to femtomolar, which is in line with previously reported data ( Figure 3B; Cha et al, 2020). However, we noticed that a longer incubation period was required for the effective detection of viral RNA molecules at low concentrations.…”

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

“…Another recently developed isothermal amplification technique is the so-called recombinase polymerase amplification (RPA) (Piepenburg et al, 2006). RPA is becoming a common choice when POC analysis is required for application in agriculture (Ahmed et al, 2018;Burkhardt et al, 2019;Strayer-Scherer et al, 2019) and forestry (Cha et al, 2020), as it presents several advantages when compared to PCR and even to LAMP. In fact, RPA does not require an initial heating step for DNA denaturation, as it exploits enzymatic activity to separate the double strand.…”

“…Moreover, the reaction temperature is quite low (37 to 42 • C), and the reaction time is usually very short (Kappagantu et al, 2017;Munawar et al, 2019). When RPA was compared to RT-LAMP as a detection method using a small (150 mm × 200 mm × 35 mm) and light (400 g) batterymounted portable optical isothermal device (Cha et al, 2020), the detection limit of RPA was shown to be 10 times lower than RT-LAMP. All these characteristics make RPA a very easy-to-use approach, especially in developing countries when fast analysis in low resource environments is needed (Wambua et al, 2017;Ghosh et al, 2018;Silva et al, 2018).…”

Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, Meloidogyne hapla, Using Recombinase Polymerase Amplification Assays

“…Real-time RPA detection assay of plant parasitic nematodes was first designed and published by Subbotin et al [ 23 ] for Meloidogyne enterolobii. RPA assays were also developed for Meloidogyne javanica , M. arenaria and M. incognita [ 24 , 25 ] and Bursaphelenchus xylophilus [ 26 , 27 ]. Recently, Song et al [ 28 ] described diagnostics of Meloidogyne hapla using RPA combined with a lateral flow dipstick assay, where species-specific primers and a probe were designed based on the effector gene 16D10 sequence.…”