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ORIGINAL ARTICLE
Keywords: Summary
Diagnosis; LAMP; Theileria annulata
A loop-mediated isothermal amplification (LAMP) assay was developed and
Correspondence: evaluated for diagnosis of tropical theileriosis. A set of six primers was
J. S. Ahmed. Division of Veterinary Infection designed based on the unique gene of Theileria annulata (Theileria annulata
Biology and Immunology, Research Center strain Ankara hypothetical protein (GeneDB TA04795). The protocol for the
Borstel, Parkallee 22, D-23845 Borstel, reaction was setup and the specificity and sensitivity of the assay were estab-
Germany. Tel.: +49 (0) 4537 188 428;
lished. The specificity experiment showed that LAMP primers amplified
Fax: +49 (0) 4537 188 627; E-mail: jahmed@
fz-borstel.de
T. annulata DNA successfully, while no amplification was seen for Theileria
parva, Theileria mutans, Theileria sergenti, Theileria sinensis, Babesia bovis as
Received for publication March 5, 2008 well as bovine genomic DNA and water control. When the sensitivity of
LAMP assay was compared with that of conventional PCR a 10-fold higher
doi:10.1111/j.1865-1682.2008.01033.x sensitivity was found, with a detection limit of 10 pg/ll of genomic DNA
isolated from a T. annulata-infected cell line. The LAMP product was con-
firmed by restriction digestion and staining with SYBR Green I. Furthermore,
the LAMP assay was applied for the diagnosis of T. annulata in field samples
and compared with reverse line blot (RLB), demonstrating that results of the
LAMP assay corresponded to those of RLB. These results indicate that the
LAMP assay is rapid and simple to run, cost-effective, sensitive and specific
and has potential usefulness for application in epidemiological studies on
T. annulata infection of cattle.
Table 1. LAMP primers designed for Theileria annulata and used in this study
poor countries, in which tropical theileriosis is seriously mediated isothermal amplification (LAMP) method for
endemic. diagnosis of equine piroplasmosis. Vet. Parasitol. 143, 155–
The analytical sensitivity of LAMP using 10-fold serial 160.
dilutions of genomic DNA isolated from a T. annulata- Ali, A. M., M. A. Bakheit, M. M. Mukhtar, S. M. Hassan,
infected cell line showed that it could detect up to 10 pg/ J. S. Ahmed, and U. Seitzer, 2006: Epidemiology of Theileria
ll. It would be of great interest to determine the sensitiv- annulata infection of dairy cattle in the Sudan using molec-
ity of LAMP in terms of parasitaemia, which needs to be ular techniques. Ann. N. Y. Acad. Sci. 1081, 471–472.
performed in the future. Nevertheless, when the sensitiv- Dolan, T. T., 1989: Theileriosis: a comprehensive review. Rev.
Sci. Tech. Off. Int. Epiz. 8, 11–36.
ity of the LAMP assay was compared with that of conven-
Grab, J. D., J. Lonsdale-Eccle, and N. Inoue, 2005: LAMP for
tional PCR, the LAMP assay demonstrated a 10-fold
tadpoles. Nat. Methods 2, 635.
higher sensitivity. An added advantage of LAMP is that in
Gubbels, J. M., A. P. de Vos, M. van der Weide, J. Viseras,
contrast to Taq DNA polymerase used in standard PCR,
L. M. Schouls, E. de Vries, and F. Jongejan, 1999: Simulta-
the Bst polymerase is not inactivated by immunoglobulin
neous detection of bovine Theileria and Babesia species by
G and many other blood components and PCR inhibitors reverse line blot hybridization. J. Clin. Microbiol. 37, 1782–
(Grab et al., 2005). Because the LAMP method shows a 1789.
high tolerance to different biological substances, determi- Ihira, M., T. Yoshikawa, Y. Enomoto, S. Akimoto, M. Ohashi,
nation of the appropriate protocol for processing the S. Suga, N. Nishimura, T. Ozaki, Y. Nishiyama, T. Notomi,
template to make it a user friendly technique, prior to Y. Ohta, and Y. Asano, 2004: Rapid diagnosis of human
large scale evaluation, is needed. herpesvirus 6 infection by a novel DNA amplification
The current study has further applied LAMP on field method, loop-mediated isothermal amplification. J. Clin.
samples collected from central Sudan. It is noteworthy Microbiol. 42, 140–145.
that results of this study do not represent valid compari- Ikadai, H., H. Tanaka, N. Shibahara, A. Matsuu, M. Uechi,
son between LAMP and RLB because of the small num- N. Itoh, S. Oshiro, N. Kudo, I. Igarashi, and T. Oyamada,
bers of samples tested. Thus, further study of the LAMP 2004: Molecular evidence of infections with Babesia gibsoni
method using a larger numbers of samples being tested parasites in Japan and evaluation of the diagnostic potential
with a reference technique such as RLB, would be of a loop-mediated isothermal amplification method. J. Clin.
required to further validate LAMP assay for application Microbiol. 42, 2465–2469.
in large scale epidemiological studies. Iwamoto, T., T. Sonobe, and K. Hayashi, 2003: Loop-
Recent studies have evaluated the diagnostic potential mediated isothermal amplification for direct detection of
of LAMP for other protozoan diseases of animals, such as Mycobacterium tuberculosis complex, M. avium, and
babesiosis (Ikadai et al., 2004; Alhassan et al., 2007). M. intracellulare in sputum samples. J. Clin. Microbiol.
41, 2616–2622.
Taken together with previous results, the results reported
Kuboki, N., N. Inoue, T. Sakurai, F. Di. Cello, D. J. Grab,
herein may indicate that LAMP could provide an accu-
H. Suzuki, C. Sugimoto, and I. Igarashi, 2003: Loop-mediated
rate, sensitive, affordable and easy-to-use method and
isothermal amplification for detection of African trypano-
practicable alternative to PCR for routine diagnosis of
somes. J. Clin. Microbiol. 41, 5517–5524.
tropical theileriosis.
Nagamine, K., K. Watanabe, K. Ohtsuka, T. Hase, and
T. Notomi, 2001: Loop-mediated isothermal amplification
Acknowledgements reaction using a nondenatured template. Clin. Chem. 47,
1742–1743.
This research was supported by the International Atomic Nagamine, K., T. Hase, and T. Notomi, 2002: Accelerated reac-
Energy Agency (IAEA) through Technical Co-operation tion by loop-mediated isothermal amplification using loop
project no. SUD/05/029 entitled ‘Characterization and primers. Mol. Cell. Probes 16, 223–229.
quality assured production of an attenuated Theileria an- Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa,
nulata vaccine’. DAS received an IAEA fellowship (SUD/ K. Watanabe, N. Amino, and T. Hase, 2000: Loop-mediated
07020). This work was supported partly by the isothermal amplification of DNA. Nucleic Acids Res. 28, e63.
EU-funded Inco-Dev project ‘INCOME’ (INCO-CT-2005- d’Oliveira, C., M. van der Weide, M. A. Habela, P. Jacquiet,
515915). and F. Jongejan, 1995: Detection of Theileria annulata in
blood samples of carrier cattle by PCR. J. Clin. Microbiol.
13, 2665–2669.
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