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A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl

Published online by Cambridge University Press:  17 October 2003

S. FERNANDEZ ,
A. H. PAGOTTO ,
M. M. FURTADO ,
Â. M. KATSUYAMA ,
A. M. B. N. MADEIRA  and
A. GRUBER
S. FERNANDEZ
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. H. PAGOTTO
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
M. M. FURTADO
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
Â. M. KATSUYAMA
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. M. B. N. MADEIRA
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. GRUBER
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
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Abstract

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1–5 pg, which corresponds approximately to 2–8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

Keywords

Eimeriadomestic fowlmultiplexpolymerase chain reactionSequence-Characterised Amplified Region (SCAR)molecular diagnosis
Type
Research Article
Information
Parasitology , Volume 127 , Issue 4 , October 2003 , pp. 317 - 325
Copyright
2003 Cambridge University Press

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Footnotes

Note: Additional figures not included in this paper are available as supplementary material at the web address http://www.lbm.fmvz.usp.br/eimeria/multiplex/

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