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The development of quantitative real-time polymerase chain reaction (PCR) assays specific to Eimeria acervulina, Eimeria maxima, Eimeria necatrix and Eimeria tenella is described and validated. The PCR templates adopted include a fragment of a gene encoding a microneme protein and previously characterized species-specific random amplified polymorphic DNA (RAPD) sequences. The sensitivity of each assay allowed the consistent detection of between one and 10 parasite genomes, equivalent to between one or two sporulated oocysts or a fraction of a single mature schizont, unaffected by the presence of chicken (host) or other Eimeria species DNA. Regression coefficients in excess of 0.99 over linear ranges of at least six orders of magnitude, together with comparable PCR efficiencies, demonstrated the robust reproducibility of each assay and suggest that two or more may be successfully multiplexed. The species-specific assays described here, combined with a previously published generic Eimeria species real-time PCR, provide valuable components in a "tool box" to accurately quantify the presence of specific Eimeria species in environmental or within-host phases of the lifecycle with little specialist knowledge. The application of these assays may benefit chicken husbandry, veterinary practice, quality control of live vaccine production and scientific research.

Original publication

DOI

10.1080/03079450701802248

Type

Journal article

Journal

Avian Pathol

Publication Date

02/2008

Volume

37

Pages

89 - 94

Keywords

Animals, Eimeria, Polymerase Chain Reaction, Reproducibility of Results, Sensitivity and Specificity, Species Specificity