A multiplex PCR assay for the simultaneous detection and discrimination of the sevenEimeriaspecies that infect domestic fowl

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.

Multiplex PCR assay using SCAR primers to detect Eimeria spp. in chicken

“…Further this aspect was incremented by the complex molecular cross interactions that took place during cycling altering the primer binding and contributing to different amplification rate of each product. The constraints due to samples with less numbers of oocysts of a particular species as observed in conventional PCR would also interfere with amplification process in multiplex PCR, all of which concurred with the opinion of Fernandez et al (2003).…”

“…Fernandez et al (2003) developed a multiplex PCR assay using SCAR species specific markers for Eimeria spp. of domestic fowl for simultaneous discrimination of various Eimeria species that infect the domestic fowl.…”

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

“…Another marker that has also been used is the sequence-characterized amplified region, which was proposed by Fernandez et al (2003b); it allows distinguishing Eimeria species in individual or in multiplex PCR. Primers that amplify fragments under the same annealing temperature are used.…”

“…For successful identification, it is necessary to have an efficient procedure for DNA extraction from oocysts; protocols with phenol/chloroform and commercial extraction kits are commonly used to break the rigid oocyst wall (Fernandez et al, 2003b;Meireles et al, 2004;Haug et al, 2007).…”

Advances in Diagnosis of Important Protozoan Diseases: Old and New Approaches

“…by using ITS-1 or ITS-2 sequences (Woods et al, 2000;Gasser et al, 2001;Lew et al, 2003;Haug et al, 2007) or sequence characterized amplification regions (SCARs) (Fernandez et al, 2003). Woods et al, (2000) described a polymerase chain reaction-linked restriction fragment length polymor-phism (PCR-RFLP) approach targeting the second internal transcribed spacer (ITS-2) to characterize six Eimeria spp.…”