A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl.
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.
Fernandez
S
S
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia-USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil.
Pagotto
A H
AH
Furtado
M M
MM
Katsuyama
A M
AM
Madeira
A M B N
AM
Gruber
A
A
D000818:Animals; D002645:Chickens; D003048:Coccidiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004539:Eimeria / Q000235:genetics* / Q000254:growth & development; D005819:Genetic Markers; D014644:Genetic Variation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D011201:Poultry Diseases / Q000469:parasitology*; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D005819:Genetic Markers