Open Access
Research (Published online: 26-11-2017)
17. Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria
A. J. Adedeji, P. A. Abdu, P. D. Luka, A. A. Owoade and T. M. Joannis
Veterinary World, 10(11): 1383-1388

A. J. Adedeji: Viral Research Division, National Veterinary Research Institute, Vom, Nigeria.
P. A. Abdu: Department of Veterinary Medicine, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria.
P. D. Luka: Biotechnology Division, National Veterinary Research Institute, Vom, Nigeria.
A. A. Owoade: Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Ibadan, Nigeria.
T. M. Joannis: Regional Laboratory for Animal Influenza and Other Transboundary Animal Diseases, National Veterinary Research Institute, Vom, Nigeria.

doi: 10.14202/vetworld.2017.1383-1388

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Article history: Received: 31-05-2017, Accepted: 24-10-2017, Published online: 26-11-2017

Corresponding author: A. J. Adedeji


Citation: Adedeji AJ, Abdu PA, Luka PD, Owoade AA, Joannis TM (2017) Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria, Veterinary World, 10(11): 1383-1388.

Aim: This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria.

Materials and Methods: HVT positive control (vaccine) was used for optimization of LAMP using six primers that target the HVT070 gene sequence of the virus. These primers can differentiate HVT, a Marek's disease virus (MDV) serotype 3 from MDV serotypes 1 and 2. Samples were collected from clinical cases of Marek's disease (MD) in chickens, processed and subjected to LAMP and PCR.

Results: LAMP assay for HVT was optimized. HVT was detected in 60% (3/5) and 100% (5/5) of the samples analyzed by PCR and LAMP, respectively. HVT was detected in the feathers, liver, skin, and spleen with average DNA purity of 3.05-4.52 μg DNA/mg (A260/A280) using LAMP. Conventional PCR detected HVT in two vaccinated and one unvaccinated chicken samples, while LAMP detected HVT in two vaccinated and three unvaccinated corresponding chicken samples. However, LAMP was a faster and simpler technique to carry out than PCR.

Conclusion: LAMP assay for the detection of HVT was optimized. LAMP and PCR detected HVT in clinical samples collected. LAMP assay can be a very good alternative to PCR for detection of HVT and other viruses. This is the first report of the use of LAMP for the detection of viruses of veterinary importance in Nigeria. LAMP should be optimized as a diagnostic and research tool for investigation of poultry diseases such as MD in Nigeria.

Keywords: herpesvirus of turkeys, loop-mediated isothermal amplification procedure, Nigeria.


1. Pastoret, P. (2004) Marek's Disease an Evolving Problem. Elsevier Ltd, London. p1-7.

2. Biggs, P.M. and Nair, V. (2012) The long view: 40 years of Marek's disease research and avian pathology. Avian Pathol., 41(1): 3-9. [Crossref] [PubMed]

3. Schat, K.A. and Nair, V. (2008) Marek's disease. In: Saif, Y.M., editor. Disease of Poultry. 12th ed. Blackwell Publishing, Iowa. p452-514.

4. Islam, T., Walkden-Brown, S.W., Renz, K.G., Fakhrul-Islam, A.F.M. and Ralapanawe, S. (2013) Vaccination-challenge interval markedly influences protection provided by Rispens CVI988 vaccine against very virulent Marek's disease virus challenge. Avian Pathol., 6: 1-11. [Crossref]

5. Morrow, C. and Fehler F. (2004) Marek's disease: A worldwide problem. Marek's Disease an Evolving Problem. Elsevier Ltd., London. p49-61. [Crossref]

6. Gimeno, I.M. (2004) Future strategies for controlling Marek's disease. In: Marek's Disease an Evolving Problem. Elsevier Ltd., London. p186-199. [Crossref]

7. Gimeno, I.M. (2008) Marek's disease vaccines: A solution for today but a worry for tomorrow? Vaccine, 26: C31-C41. [Crossref] [PubMed]

8. Atkins, K.E., Read, A.F., Savill, N.J., Renz, K.G., Islam, A.F.M., Walkden-Brown, S.W. and Woolhouse, M.E.J. (2013) Vaccination and reduced cohort duration can drive virulence evolution: Mareks disease virus and industrialized agriculture. Evolution, 67: 851-860. [Crossref] [PubMed]

9. Zelnik, V. (2004) Diagnosis of Marek's disease In: Marek's Disease an Evolving Problem. Elsevier Ltd, London. p156-167. [Crossref]

10. Cortes, A.L., Montiel, E.R., Lemiere, S. and Gimeno, I.M. (2011) Comparison of blood and feather pulp samples for the diagnosis of Marek's disease and for monitoring Marek's disease vaccination by real time-PCR. Avian Dis., 55: 302-310. [Crossref] [PubMed]

11. Wozniakowski, G., Samorek-Salamonowicz, E. and Kozdrun, W. (2011) Rapid detection of Marek's disease virus in feather follicles by loop-mediated isothermal amplification. Avian Dis., 55: 462-467. [Crossref] [PubMed]

12. Pestana, E.A., Belak, S., Diallo, A., Crowther, J.R. and Viljoen, G.J. (2010) Early, Rapid and Sensitive Veterinary Molecular Diagnostics Real Time PCR Applications. International Atomic Energy Agency and Springer, Dordrecht, Netherlands. p1-319. [Crossref]

13. Notomi, T., Okayama, H., Yonekawa, T., Watana, K., Amino, N. and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res., 28: 63-68. [Crossref]

14. Nawathe, D.R., Ojeh, C.K. and Onunkwo, O. (1978) Incidence of Marek's disease in Northern states of Nigeria. Vet. Rec., 102: 128. [Crossref] [PubMed]

15. Fatunmbi, O.O. and Adene, D.F. (1986) A ten year prevalence study of Marek's disease and avian leukoses at Ibadan, Nigeria. Acta Vet., 55: 49-53. [Crossref]

16. Owoade, A.A. and Oni, O.O. (2008) Molecular detection of Marek's disease virus in some poultry flocks in southwestern Nigeria by polymerase chain reaction. Niger. Poult. Sci. J., 5(3): 113-118.

17. Wakawa, A.M., Muhammad, Z.K., Aliyu, H.B. and Mohammed, B. (2012) A retrospective analysis of Marek's disease diagnosed at poultry clinic of Ahmadu Bello university, Zaria, Nigeria. J. Vet. Adv., 2(8): 424-429.

18. Jwander, L.D., Abdu, P.A., Ibrahim, N.D.G. and Nok, A.J. (2014) Retrospective studies of Marek's disease diagnosed in four States across Nigeria. J. Vet. Adv., 4(6): 556-563.

19. Dunn, J.R. and Gimeno, I.M. (2013) Current status of Marek's disease in the United States and worldwide based on a questionnaire survey. Avian Dis., 57: 483-490. [Crossref] [PubMed]

20. Woz'niakowski, G., Samorek-Salamonowicz, E. and Kozdrun, W. (2013) Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotype 1, 2 and 3. Avian Dis., 57: 1-5.

21. Xu, J., Zhang, Z., Yin, Y., Cui, S., Xu, S., Guo, Y., Li, J., Wang, J., Liu, X. and Han, L. (2009) Development of reverse-transcription loop-mediated isothermal amplification for the detection of infectious bursal disease virus. J. Virol. Methods, 162: 267-271. [Crossref] [PubMed]

22. Deng, X., Qi, X., Yulong, G.Y., Wang, Y., Qin, L., Gao, H., Gao, L. and Wang, X. (2010) Development of a loop-mediated isothermal amplification method for rapid detection of reticuloendotheliosisvirus. J. Virol. Methods, 168: 82-86. [Crossref] [PubMed]

23. Li, L., Bao, J., Wu, X., Wang, Z., Wang, J., Gong, M., Liu, C. and Li, J. (2010) Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay. J. Virol. Methods, 170: 37-41. [Crossref] [PubMed]

24. Wang, Y., Kang, Z., Gao, Y., Qin, L., Chen, L., Wang, Q., Li, J., Gao, H., Qi, X., Lin, H. and Wang, X. (2011) Development of loop-mediated isothermal amplification for rapid detection of avian Leukosis virus subgroup A. J. Virol. Methods, 173: 31-36. [Crossref] [PubMed]

25. Angamuthu, R., Baskaran, S., Gopal, D.R., Devarajan, J. and Kathaperumal, K. (2012) Rapid detection of the Marek's disease viral genome in chicken feathers by loop-mediated isothermal amplification. J. Clin. Microbiol., 50(3): 961-965. [Crossref] [PubMed] [PMC]

26. Yamazaki, W., Mioulet, V., Murray, L., Madi, M., Haga, T., Misawa, N., Horii, Y. and King, P.D. (2013) Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus. J. Virol. Methods, 192: 18-24. [Crossref]

27. Wei, X., Xingming, S., Yan, Z., Jing, Z., Mei, W., Changjun, L., Hongyu, C., Shunlei, H., Yanming, Q., Hongyan, C. and Yunfeng, W. (2012) Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meqgene. J. Virol. Methods, 183(2): 196-200. [Crossref] [PubMed]

28. Wozniakowski, G. and Niczyporuk, J.S. (2015) Detection of specific UL49 sequences of Marek's diseasevirus CVI988/rispens strain using loop-mediated isothermal amplification. J. Virol. Methods, 221: 22-28. [Crossref] [PubMed]

29. Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Rock, D.L. and Kutish, G.F. (2001) The genome of turkey herpesvirus. J. Virol., 1: 971-978. [Crossref] [PubMed] [PMC]

30. Le Gros, F.X., Dancer, A., Giacomini, C., Pizzoni, L., Bublot, M., Graziani, M. and Prandini, F. (2009) Field efficacy trial of a novel HVT-IBD vector vaccine for 1-day-old broilers. Vaccine, 27: 592-596. [Crossref] [PubMed]

31. Rauw, F., Gardin Y., Palya, V., Anbari, S., Lemaire, S., Boschmans M., van den Berg, T., and Lambrecht, B. (2010) Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine, 28: 823-833. [Crossref] [PubMed]

32. Prandini, F., Simon, B., Jung, A., Poppel, M., Lemiere, S. and Rautenschlein, S. (2016) Comparison of infectious bursal disease live vaccines and a HVT-IBD vector vaccine and their effects on the immune system of commercial layer pullets. Avian Pathol., 45(1): 114-125. [Crossref] [PubMed]

33. Walkden-Brown, S.W., Islam, A., Groves, P.J., Rubite, A., Sharpe, S.M. and Burgess, S.K. (2013) Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR. Avian Dis., 57: 544-554. [Crossref]

34. Couteaudier, M. and Denesvre, C. (2014) Marek's disease virus and skin interactions. Vet. Res., 45: 36. [Crossref] [PubMed] [PMC]