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

Autoři: Deokjea Cha aff001;  Dongsoo Kim aff001;  Wonil Choi aff002;  Sungjun Park aff003;  Hyerim Han aff001
Působiště autorů: Division of Forest Insect Pests & Diseases, National Institute of Forest Science, Dongdaemun, Seoul, Korea aff001;  Division of Forest Ecology & Climate Change, National Institute of Forest Science, Dongdaemun, Seoul, Korea aff002;  2NCBio, Seo-gu, Daejeon, Korea aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227476


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.

Klíčová slova:

Forests – Nematode infections – Optical equipment – Pines – Polymerase chain reaction – Recombinase polymerase amplification – Sequence alignment – Trees


1. Klapwijk MJ, Hopkins AJM, Eriksson L, Pettersson M, Schroeder M, Lindelöw Å, et al. Reducing the risk of invasive forest pests and pathogens: Combining legislation, targeted management and public awareness. Ambio. 2016;45: 223–234. doi: 10.1007/s13280-015-0748-3 26744056

2. Vander Zanden MJ, Hansen GJA, Higgins SN, Kornis MS. A pound of prevention, plus a pound of cure: Early detection and eradication of invasive species in the Laurentian Great Lakes. J Great Lakes Res. 2010;36: 199–205. doi: 10.1016/j.jglr.2009.11.002

3. Linit MJ. Nematode-vector relationships in the pine wilt disease system. J Nematol. 1988;20: 227–235. 19290206

4. Li H, Trinh PQ, Waeyenberge L, Moens M. Characterisation of Bursaphelenchus spp. isolated from packaging wood imported at Nanjing, China. Nematology. 2009;11: 375–408. doi: 10.1163/156854109X446971

5. Futai K. Role of asymptomatic carrier trees in epidemic spread of pine wilt disease. J For Res. 2003;8: 253–260. doi: 10.1007/s10310-003-0034-2

6. Mota MM, Braasch H, Bravo MA, Penas AC, Burgermeister W, Metge K, et al. First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology. 1999;1: 727–734. doi: 10.1163/156854199508757

7. Robertson L, Cobacho Arcos S, Escuer M, Santiago Merino R, Esparrago G, Abelleira A, et al. Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain. Nematology. 2011;13: 755–757. doi: 10.1163/138855411X578888

8. Cheng H, Lin M, Li W, Fang Z. The occurrence of a pine wilting disease caused by a nematode found in Nanjing. For Pest Dis. 1983;4: 1–5.

9. Tzean S, Jan S. The occurrence of pine wood nematode, Bursaphelenchus xylophilus, in Taiwan. Proceedings of the 6th ROC Symposium of Electron Microscopy. 1985. pp. 38–39.

10. Yi C, Byun B, Park J, Yang S, Chang KH. First finding of the pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle and its insect vector in Korea. Res Reports For Res Inst. 1989;38: 141–149.

11. Hu S, Ning T, Fu D, Haack RA, Zhang Z, Chen D, et al. Dispersal of the Japanese Pine Sawyer, Monochamus alternatus (Coleoptera: Cerambycidae), in Mainland China as inferred from molecular data and associations to indices of human activity. PLoS One. 2013;8: e57568. doi: 10.1371/journal.pone.0057568 23469026

12. Hirata A, Nakamura K, Nakao K, Kominami Y, Tanaka N, Ohashi H, et al. Potential distribution of pine wilt disease under future climate change scenarios. PLoS One. 2017;12: e0182837. doi: 10.1371/journal.pone.0182837 28797067

13. Hooper D, Evans K. Extraction, identification and control of plant parasitic nematodes. In: Evans K, Trudgill D, Webster J, editors. Plant parasitic nematodes in temperate agriculture. CABI; 1993. pp. 1–60.

14. Braasch H, Burgermeister W, Gu J. Revised intra-generic grouping of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae). J Nematode Morphol Syst. 2009;12: 65–88.

15. Iwahori H, Kanzaki N, Futai K. A simple, polymerase chain reaction-restriction fragment length polymorphism-aided diagnosis method for pine wilt disease. Forest Pathology. 2000. pp. 157–164. doi: 10.1046/j.1439-0329.2000.00201.x

16. Kikuchi T, Aikawa T, Oeda Y, Karim N, Kanzaki N. A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology. 2009;99: 1365–1369. doi: 10.1094/PHYTO-99-12-1365 19900002

17. Takeuchi Y, Kanzaki N, Futai K. A nested PCR-based method for detecting the pine wood nematode, Bursaphelenchus xylophilus, from pine wood. Nematology. 2005;7: 775–782. doi: 10.1163/156854105775142928

18. Kang JS, Kim A-Y, Han HR, Moon YS, Koh YH. Development of two alternative Loop-mediated isothermal amplification tools for detecting pathogenic pine wood nematodes. For Pathol. 2015;45: 127–133.

19. Leal I, Allen E, Humble L, Green M, Rott M. Application of conventional PCR and real-time PCR diagnostic methods for detection of the pinewood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine. Pine Wilt Dis A Worldw Threat to For Ecosyst. 2007;9: 351–362. doi: 10.1007/978-1-4020-8455-3_17

20. Cha DJ, Kim DS, Lee SK, Han HR. A new on-site detection method for Bursaphelenchus xylophilus in infected pine trees. For Pathol. 2019;49: e12503.

21. Cardoso JMS, Fonseca L, Abrantes I. Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector. Eur J Plant Pathol. 2012;133: 419–425. doi: 10.1007/s10658-011-9915-y

22. Takeuchi Y, Futai K. Asymptomatic carrier trees in pine stands naturally infected with Bursaphelenchus xylophilus. Nematology. 2007;9: 243–250. doi: 10.1163/156854107780739027

23. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008;3: 877–882. doi: 10.1038/nprot.2008.57 18451795

24. Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006;4: e204. doi: 10.1371/journal.pbio.0040204 16756388

25. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012;113: 1014–1026. doi: 10.1111/j.1365-2672.2012.05384.x 22747964

26. Leal I, Allen E, Foord B, Anema J, Reisle C, Uzunovic A, et al. Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). For Pathol. 2015;45: 134–148.

27. Bass D, Stentiford GD, Littlewood DTJ, Hartikainen H. Diverse Applications of Environmental DNA Methods in Parasitology. Trends Parasitol. 2015;31: 499–513. doi: 10.1016/j.pt.2015.06.013 26433253

28. Sato MO, Rafalimanantsoa A, Ramarokoto C, Rahetilahy AM, Ravoniarimbinina P, Kawai S, et al. Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar. Int J Infect Dis. 2018;76: 130–136. doi: 10.1016/j.ijid.2018.08.018 30201503

29. Thomas AC, Deagle BE, Eveson JP, Harsch CH, Trites AW. Quantitative DNA metabarcoding: Improved estimates of species proportional biomass using correction factors derived from control material. Mol Ecol Resour. 2016;16: 714–726. doi: 10.1111/1755-0998.12490 26602877

30. Hoover K, Uzunovic A, Gething B, Dale A, Leung K, Ostiguy N, et al. Lethal temperature for pinewood nematode, Bursaphelenchus xylophilus, in infested wood using microwave energy. J Nematol. 2010;42: 101–110. 22736846

Článek vyšel v časopise


2020 Číslo 1