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A new PCR-RFLP method for detection of Anaplasma marginale based on 16S rRNA

Veterinary Research Communications volume 34, pages 43–50 (2010)Cite this article

Abstract

Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are obligate intracellular tick borne pathogens. Three species of Anaplasma that infect cattle and sheep (A. marginale, A. centrale and A. ovis) are well recognized. Of these erythrocytic Anaplasma, A. marginale can cause diseases in the livestock with high economical losses. Species-specific PCR based on 16S rRNA gene is commonly used for detection of Anaplasma species but can not differentiate A. marginale, A. centrale and A. ovis because of sequence similarity. In this study DNA extraction was performed on 50 blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells was analyzed by PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I only recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it. The nucleotide sequence of the A. marginale 16S rRNA gene was determined and compared with the sequences of A. marginale in GenBank. The 16S rRNA of A. marginale in Iran was completely similar to the related sequence deposited in GenBank at accession number of M60313. In the present study we propose a new PCR-restriction fragment length polymorphism analysis (RFLP) method based on 16S rRNA gene for specific detection of A. marginale.

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Fig. 1

Abbreviations

PCR:

polymerase chain reaction

RFLP:

restriction fragment length polymorphism

OD:

optical density

RLB:

Reverse Line Blot

TBE:

Tris-Borate-EDTA

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Acknowledgements

This work was supported by Grand no. 7502016/6/2 of University of Tehran, Investigating Group Molecular Biological System Transfer (MBST) in Iran and Veterinary department of Isfahan research center for agriculture and natural resources. The authors declare that there is no conflict of interests.

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Authors and Affiliations

  1. Veterinary Department of Isfahan Research Center for Agriculture and Natural Resources, Isfahan, Iran

    Vahid Noaman

  2. Center for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

    Parviz Shayan

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  1. Vahid Noaman
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  2. Parviz Shayan
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Correspondence to Vahid Noaman.

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Noaman, V., Shayan, P. A new PCR-RFLP method for detection of Anaplasma marginale based on 16S rRNA. Vet Res Commun 34, 43–50 (2010). https://doi.org/10.1007/s11259-009-9331-3

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Keywords

  • Anaplasma marginale
  • 16S rRNA gene
  • PCR-RFLP