Experimental and Applied Acarology

, Volume 77, Issue 1, pp 65–72 | Cite as

Development of a loop-mediated isothermal amplification (LAMP) assay for the detection of Anaplasma marginale

  • Rodrigo GigliotiEmail author
  • César Cristiano Bassetto
  • Cintia Hiromi Okino
  • Henrique Nunes de Oliveira
  • Márcia Cristina de Sena Oliveira


Parasitemia generated by Anaplasma marginale causes significant losses in the cattle industry. A major constraint to the effective control and management of anaplasmosis in livestock is the lack of a rapid and reliable diagnostic test to identify the parasite and allow for immediate therapy. In the present study, we developed a novel DNA-based assay for the detection of A. marginale in bovine blood samples, using loop-mediated isothermal amplification (LAMP). DNA from six cattle and hemoparasite samples (Babesia bovis, Babesia bigemina, Anaplasma centrale and A. marginale) were tested for specificity, sensitivity and cross-reactions. The developed LAMP procedures were also confirmed and compared with the qPCR method. The same gene sequence (major surface protein 1b, msp1b) of A. marginale was used to design a set of primers for the LAMP and qPCR assays. The results showed that LAMP is specific, as no positive signal was observed for the other tested hemoparasites. However, the sensitivity of the qPCR assay was ten times higher than LAMP. Our findings indicate that this LAMP method has a good sensitivity and high specificity for the detection of A. marginale and may have a potential application in the detection and differentiation of bovine anaplasmosis.


LAMP qPCR Anaplasma marginale Detection Sensibility Specificity 



This research project was supported by the São Paulo State Research Support Foundation (FAPESP) (Grant # 2016/07216-7, 2017/11297-5), the National Council for Scientific and Technological Development (CNPq) (Grant # 153231/2018-1) and the Brazilian Agricultural Research Corporation (Embrapa SEG


  1. Alhassan A, Thekisoe OMM, Yokoyama N, Inoue N, Motloang MY, Mbati PA, Yin H, Katayama Y, Anzai T, Sugimoto C, Igarashi I (2007) Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis. Vet Parasitol 143:155–160CrossRefGoogle Scholar
  2. Carelli G, Decaro N, Lorusso A, Elia G, Lorusso E, Mari V, Ceci L, Buonavoglia C (2007) Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR Vet. Microbiol 124(1–2):107–114Google Scholar
  3. Giglioti R, Oliveira HN, Bilhassi TB, Portilho AI, Okino CH, Marcondes CR, Oliveira MCS (2018) Estimates of repeatability and correlations of hemoparasites infection levels for cattle reared in endemic areas for Rhipicephalus microplus. Vet Parasitol 250:78–84CrossRefGoogle Scholar
  4. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K-I (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxyl naphthol blue. Biotechniques 46:167–172CrossRefGoogle Scholar
  5. Ikadai H, Tanaka H, Shibahara N, Matsuu A, Uechi M, Itoh N, Oshiro S, Kudo N, Igarashi I, Oyamada T (2004) Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol 42(6):2465–2469CrossRefGoogle Scholar
  6. Iseki H, Alhassan A, Ohta N, Thekisoe OMM, Yokoyama N, Inoue N, Nambota A, Yasuda J, Igarashi I (2007) Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods 71:281–287CrossRefGoogle Scholar
  7. Kocan KM, de la Fuente J, Blouin EF, Coetzee JF, Ewing SA (2010) The natural history of Anaplasma marginale. Vet Parasitol 167:95–107CrossRefGoogle Scholar
  8. Liu A, Guan G, Du P, Gou H, Liu Z, Liu J, Ma M, Yang J, Li Y, Niu Q, Ren Q, Bai Q, Yin H, Luo J (2012) Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis. and B. bigemina. Parasitol Int 61:658–663CrossRefGoogle Scholar
  9. Liu J, Xu L, Guo J, Chen R, Grisham MP, Que Y (2013) Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane. Biomed Res Int. Google Scholar
  10. Ma M, Liu Z, Sun M, Yang J, Guan G, Li Y, Luo J, Yin H (2011) Development and evaluation of a loop-mediated isothermal amplification method for rapid detection of Anaplasma ovis. J Clin Microbiol 49(6):2143–2146CrossRefGoogle Scholar
  11. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154CrossRefGoogle Scholar
  12. Noden BH, Martin J, Carrillo Y, Talley JL, Ochoa-Corona FM (2018) Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia. PLoS One 13(2):e0192331CrossRefGoogle Scholar
  13. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63CrossRefGoogle Scholar
  14. Notomi T, Mori Y, Tomita N, Kanda H (2015) Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. J Microbiol 53:1–5CrossRefGoogle Scholar
  15. 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 3:877–882CrossRefGoogle Scholar
  16. Wang X, Seo DJ, Lee MW, Choi C (2014) Comparison of conventional PCR, Multiplex PCR, and loop mediated isothermal amplification assays for rapid detection of Arcobacter species. J Clin Microbiol 52(2):557–563CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita FilhoJaboticabalBrazil
  2. 2.Embrapa Pecuária SudesteSão CarlosBrazil
  3. 3.Centro de Pesquisa de Genética e Reprodução Animal, Instituto de ZootecniaNova OdessaBrazil

Personalised recommendations