Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples

Particle and Fibre Toxicology

Jean-Luc Gala⁵  Bertrand Losson¹  Bernard Mignon¹  Jean-Pierre Doucet⁶  Catherine Dumont³  Renata Fogt-Wyrwas²  Leonid M Irenge⁵  Jean-Francois Durant⁴ 

[1] Département des Maladies Infectieuses et Parasitaires, Faculté de Médecine Vétérinaire, Université de Liège (Ulg), boulevard de Colonster, 20, B43, 4000, Liège, Belgium;Department of Biology and Environmental Protection, University School of Physical Education, Królowej Jadwigi 27/39, 61-871, Poznań, Poland;Royal Military Academy, Avenue de la Renaissance 30, 1000, Bruxelles, Belgium;Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Clos chapelle-aux-champs, 30 B1.30.24, 1200, Brussels, Belgium;Defense Laboratories Department, ACOS Ops&Trg, Belgian Armed Forces, Martelarenstraat, 181, 1800, Peutie, Belgium;Clinivet, clinique vétérinaire de Gosselies, rue pont à Migneloux 39, 6041, Gosselies, Belgium

关键词: Samples;    Soil;    Fecal;    Eggs;    Toxocara;    ITS2;    Duplex real-time PCR;   

Others  :  1228812
DOI  :  10.1186/1756-3305-5-288

 received in 2012-08-24, accepted in 2012-11-23,  发布年份 2012

【 摘 要 】

Background

Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount.

Methods

A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination.

Results

2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces.

Conclusion

The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

【 授权许可】

   
2012 Durant et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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