A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples

Particle and Fibre Toxicology

Mikael Juremalm⁷  Peter Deplazes⁶  Adriano Casulli⁴  Morten Lukacs³  Anders Holmberg³  Andrea Miller¹  Erik Olof Ågren²  Helene Wahlström⁵  Maria Teresa Armua-Fernandez⁶  Åsa Hagström⁷  Mats Isaksson⁷ 

[1] Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden;Department of Pathology and Wildlife Diseases, National Veterinary Institute, Uppsala, Sweden;Precision System Science, Stockholm, Sweden;Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy;Department of Epidemiology, National Veterinary Institute, Uppsala, Sweden;Institute of Parasitology, Vetsuisse and Medical Faculty, University of Zurich, Zurich, Switzerland;Department of Virology Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden

关键词: Faecal samples;    Real-time PCR;    Magnetic capture;    Diagnostic method;    Red fox;    Surveillance;    Echinococcus multilocularis;   

Others  :  1148326
DOI  :  10.1186/s13071-014-0583-6

 received in 2014-06-04, accepted in 2014-11-30,  发布年份 2014

【 摘 要 】

Background

Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012–2013 on more than 2000 faecal samples.

Methods

We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic.

Results

Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR.

The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7 -100).

Conclusions

The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

【 授权许可】

   
2014 Isaksson et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

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