, Volume 74, Issue 5, pp 477–485 | Cite as

Loop-mediated isothermal amplification combined with PCR for specific identification of injurious mite, Tetranychus urticae (Trombidiformes: Tetranychidae)

  • Samira Sinaie
  • Hussein Sadeghi-NamaghiEmail author
  • Lida Fekrat
Original Article


Identification of spider mites of the genus Tetranychus is strenuous due to their minute size and the restricted number of diagnostic characters. Most species of the Iranian Tetranychus spider mites are morphologically alike, distinguishable only based on male aedeagus; however, most of the intercepted Tetranychus specimens are females, leaving their identification at the genus level. Tetranychus urticae is a highly polyphytophagous pest species commonly found in greenhouses of Iran. As the unambiguous identification of a pest is the first crucial step toward implementing any effective pest management procedure, various DNA-based methods have formerly been used as complement to the traditional morphological methods. In the current study, a technique integrating loop-mediated isothermal amplification (LAMP) with PCR (polymerase chain reaction) was utilized for quick detection of T. urticae and discriminating it from other Tetranychus species attacking greenhouse crops. The assay was validated against various developmental stages of T. urticae specimens. Not only did the PCR-LAMP assay decrease the time needed for identification, but also was species-specific and sensitive enough to be applied even by non-specialists.


Two-spotted spider mite Tetranychinae Major pest Molecular diagnosis LAMP 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11756_2018_187_MOESM1_ESM.pdf (110 kb)
ESM 1 (PDF 109 kb)
11756_2018_187_MOESM2_ESM.pdf (297 kb)
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11756_2018_187_MOESM3_ESM.pdf (197 kb)
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  1. Arimoto M, Satoh M, Uesugi R, Osakabe M (2013) PCR-RFLP analysis for identification of Tetranychus spider mite species (Acari: Tetranychidae). J Econ Entomol 106(2):661–668. CrossRefGoogle Scholar
  2. Ben-David T, Melamed S, Gerson U, Morin S (2007) ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae). Exp Appl Acarol 41(3):169–181. CrossRefGoogle Scholar
  3. Bonizzoni M, Afrane Y, Yan GY (2009) Loop-mediated isothermal amplification (LAMP) for rapid identification of Anopheles gambiae and Anopheles arabiensis mosquitoes. Am J Trop Med Hyg 81:1030–1034. CrossRefGoogle Scholar
  4. Da Silva Gonçalves D, Cassimiro APA, de Oliveira CD, Rodrigues NB, Moreira LA (2014) Wolbachia detection in insects through LAMP: loop mediated isothermal amplification. Parasit Vectors 7(1):228 CrossRefGoogle Scholar
  5. De Mendonça RS, Navia D, Diniz IR, Auger P, Navajas M (2011) A critical review on some closely related species of Tetranychus sensu stricto (Acari: Tetranychidae) in the public DNA sequences databases. Exp Appl Acarol 55(1):1–23. CrossRefGoogle Scholar
  6. Ehara S (1999) Revision of the spider mite family Tetranychidae of Japan (Acari: Prostigmata). Species Divers 4(1):63–141. CrossRefGoogle Scholar
  7. Faggion SA, Salvador AR, Jacobino KL, Bortolotto LFB, Lopes MB, Silva M, Santos EV, Fachin AL, França SC, Marins M (2013) Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs. Arch Med Vet 45(2):197–201 CrossRefGoogle Scholar
  8. Fekrat L, Zakiaghl M, Tahan V (2015) Application of the LAMP assay as a diagnostic technique for rapid identification of Thrips tabaci (Thysanoptera: Thripidae). J Econ Entomol 108(3):1337–1343. CrossRefGoogle Scholar
  9. Fournier D, Bride JM, Navajas M (1994) Mitochondrial DNA from a spider mite: isolation, restriction map and partial sequence of the cytochrome oxidase subunit I gene. Genetica 94(1):73–75. CrossRefGoogle Scholar
  10. Fukuta S, Kato S, Yoshida K, Mizukami Y, Ishida A, Ueda J et al (2003) Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J Virol Methods 112(1–2):35–40. CrossRefGoogle Scholar
  11. Fukuta S, Ohishi K, Yoshida K, Mizukami Y, Ishida A, Kanbe M (2004) Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum. J Virol Methods 121:49–55. CrossRefGoogle Scholar
  12. Gotoh T, Araki R, Boubou A, Migeon A, Ferragut F, Navajas M (2009) Evidence of co-specificity between Tetranychus evansi and Tetranychus takafujii (Acari: Prostigmata, Tetranychidae): comments on taxonomic and agricultural aspects. Int J Acarol 35(6):485–501. CrossRefGoogle Scholar
  13. Grbić M, Van Leeuwen T, Clark RM, Rombauts S, Rouze P, Whistlecraft J et al (2011) The genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature 479:487–492. CrossRefGoogle Scholar
  14. Guan X, Guo J, Shen P, Yang L, Zhang D (2010) Visual and rapid detection of two genetically modified soybean events using loop-mediated isothermal amplification method. Food Anal Methods 3(4):313–320. CrossRefGoogle Scholar
  15. Hinomoto N, Tran DP, Pham AT, Le TBN, Tajima R, Ohashi K, Osakabe M, Takafuji A (2007) Identification of spider mites (Acari: Tetranychidae) by DNA sequences: a case study in Northern Vietnam. Int J Acarol 33(1):53–60. CrossRefGoogle Scholar
  16. Hirayama H, Kageyama S, Moriyasu S, Sawai K, Onoe S, Takahashi Y, Katagiri S, Toen K, Watanabe K, Notomi T, Yamashina H, Matsuzaki S, Minamihashi A (2004) Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification. Theriogenology 62(5):887–896. CrossRefGoogle Scholar
  17. Hsieh CH, Wang HY, Chenc YF, Koa CC (2012) Loop-mediated isothermal amplification for rapid identification of biotypes B and Q of the globally invasive pest Bemisia tabaci, and studying population dynamics. Pest Manag Sci 68(8):1206–1213. CrossRefGoogle Scholar
  18. Huang CG, Hsu JC, Haymer DS, Lin GC, Wu WJ (2009) Rapid identification of the Mediterranean fruit fly (Diptera: Tephritidae) by loop-mediated isothermal amplification. J Econ Entomol 102(3):1239–1246. CrossRefGoogle Scholar
  19. Itakura S, Kankawa T, Tanaka H, Enoki A (2006) Identification of two subterranean termite species (Isoptera: Rhinotermitidae) using the loop-mediated isothermal amplification (LAMP) method. Sociobiology 47:99–113Google Scholar
  20. Iwamoto T, Sonobe T, Hayashi K (2003) Loop mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol 41(6):2616–2622. CrossRefGoogle Scholar
  21. Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI (2017) FastPCR: an in silico tool for fast primer and probe design and advanced sequence analysis. Genomics 109(4–5):312–319. CrossRefGoogle Scholar
  22. Kitano T, Mikami Y, Iwase T, Asano M, Komiyama K (2016) Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis. J Oral Sci 58(2):163–169. CrossRefGoogle Scholar
  23. Kuan CP, Wu MT, Lu YL, Huang HC (2010) Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification. J Virol Methods 169(1):61–65. CrossRefGoogle Scholar
  24. Li D, Fan Q-H, Waite DW, Gunawardana D, George S, Kumarasinghe L (2015) Development and validation of a real-time PCR assay for rapid detection of two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae). PLoS One 10(7):e0131887. CrossRefGoogle Scholar
  25. Li F, Yan W, Long L, Qi X, Li C, Zhang S (2014) Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops. Int J Mol Sci 15(9):15109–15121. CrossRefGoogle Scholar
  26. Matsuda T, Fukumoto C, Hinomoto N, Gotoh T (2013) DNA-based identification of spider mites: molecular evidence for cryptic species of the genus Tetranychus (Acari: Tetranychidae). J Econ Entomol 106(1):463–472. CrossRefGoogle Scholar
  27. Matsuda T, Hinomoto N, Singh RN, Gotoh T (2012) Molecular based identification and molecular phylogeny of Oligonychus species (Acari: Tetranychidae). J Econ Entomol 105(3):1043–1050. CrossRefGoogle Scholar
  28. Matsuda T, Morishita M, Hinomoto N, Gotoh T (2014) Phylogenetic analysis of the spider mite sub-family Tetranychinae (Acari: Tetranychidae) based on the mitochondrial COI gene and the 18S and the 5′ end of the 28S rRNA genes indicates that several genera are polyphyletic. PLoS One 9(10):e108672. CrossRefGoogle Scholar
  29. Navajas M, Boursot P (2003) Nuclear ribosomal DNA monophyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive. Proc Biol Sci 270(Suppl 1):S124–S127. Google Scholar
  30. Notomi T, Okayma H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):e63. CrossRefGoogle Scholar
  31. Ohtsuki R, Kawamoto K, Kato Y, Shah MM, Ezaki T, Makino SI (2008) Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method. J Appl Microbiol 104(6):1815–1823. CrossRefGoogle Scholar
  32. Osakabe M, Hirose T, Sato M (2002) Discrimination of four Japanese Tetranychus species (Acari: Tetranychidae) using PCR-RFLP of the inter-transcribed spacer region of nuclear ribosomal DNA. Appl Entomol Zool 37(3):399–407. CrossRefGoogle Scholar
  33. Osakabe M, Kotsubo Y, Tajima R, Hinomoto N (2008) Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae). J Econ Entomol 101(4):1167–1175. CrossRefGoogle Scholar
  34. Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, Islam MA, Inoue S, Hosaka N, Morita K (2005) Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 43(6):2895–2903. CrossRefGoogle Scholar
  35. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52(2):303–306. CrossRefGoogle Scholar
  36. Ravindran A, Levy J, Pierson E, Gross DC (2012) Development of a loop mediated isothermal amplification procedure as a sensitive and rapid method for detection of Candidatus liberibacter solanacearum in potatoes and psyllids. Phytopathology 102(9):899–907.
  37. Sabahi S, Fekrat L, Zakiaghl M, Moravej GH (2017) Loop-mediated isothermal amplification combined with PCR for rapid identification of the Ethiopian fruit fly (Diptera: Tephritidae). Neotrop Entomol 47(1):96–105. CrossRefGoogle Scholar
  38. Sabelis MW (1991) Life-history evolution of spider mites. In: The Acari: reproduction, development and life-history strategies (chapter 2). Chapman and Hall, London, pp 23–50. CrossRefGoogle Scholar
  39. Thai HTC, Le MQ, Vuong CD, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K (2004) Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 42:1956–1961. CrossRefGoogle Scholar
  40. Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3(5):877–882. CrossRefGoogle Scholar
  41. Wauthy G, Noti MI, Leponce M, Bauchau V (1998) Taxy and variations of leg setae and solenidia in Tetranychus urticae (Acari, Prostigmata). Acarologia 39(3):233–255Google Scholar
  42. Xu J, Zheng Q, Yu L, Liu R, Zhao X, Wang G, Wang Q, Cao J (2013) Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25. Food Sci Nutr 1(6):432–438. CrossRefGoogle Scholar
  43. Zhang ZQ, Jacobson RJ (2000) Using adult female morphological characters for differentiating Tetranychus urticae complex (Acari: Tetranychidae) from greenhouse tomato crops in UK. Syst Appl Acarol 5(1):69–76. Google Scholar

Copyright information

© Institute of Zoology, Slovak Academy of Sciences 2019

Authors and Affiliations

  • Samira Sinaie
    • 1
  • Hussein Sadeghi-Namaghi
    • 1
    Email author
  • Lida Fekrat
    • 1
  1. 1.Department of Plant Protection, Faculty of AgricultureFerdowsi University of MashhadMashhadIran

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