Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA

Abstract

SUMMARY: A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers forEchinococcus multilocularis(amplicon size 395 bp) were species-specific as assessed byin silicoanalysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA fromE. granulosusorTaeniaspp. was not possible. The primers designed forE. granulosusalso amplified DNA (117 bp) fromE. vogeli, and those designed forTaeniaspp. amplified products (267 bp) from species ofMesocestoides,DipylidiumandDiphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the describedE. granulosusgenotypes.Taeniaspp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.