Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis - PubMed (original) (raw)

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

Jenny Knapp et al. Appl Environ Microbiol. 2016.

Abstract

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1

FIG 1

Sequence alignment (partial cox1 gene) of 111-bp sequences from Toxocara_cati_ and T. canis reference specimens, adult worms from the laboratory collection (T. canis, 9 specimens; and T. cati, 2 specimens), and three positive samples from the present coprosample collection.

FIG 2

FIG 2

Box plots representing the results of Toxocara qPCR performed on DNA extracted from isolated T. cati eggs. Results are expressed as numbers of quantitative cycles (Cq); all qPCRs were performed in triplicate.

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