In Europe the red fox Vulpes vulpes serves as the

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In Europe, the red fox (Vulpes vulpes) serves as the main final host and small rodents such as Microtus spp., Arvicola spp. and Myodes spp. are the main intermediate hosts (Woolsey et al., 2015; Woolsey et al., 2016). Other canids and small rodent species can also contribute to the maintenance of the E. multilocularis <a  href='http://www.sulfo-nhs-biotin.com/?p=11219'>life compound library</a>  (Kapel et al., 2006). Intermediate and aberrant hosts (e.g. humans, monkeys, pigs) get infected orally, through the uptake of infectious eggs shed by the final hosts with the faeces (Bottcher et al., 2013; Brack et al., 1997; Woolsey et al., 2016).<br />E. multilocularis infection in final hosts is usually detected post mortem using the Sedimentation and Counting Technique (SCT), which is regarded as the gold standard among the laboratory techniques (Eckert, 2003; Eckert et al., 2001), the Intestinal Scraping Technique (IST), or variants of these methods (Tackmann et al., 2006; Umhang et al., 2011). Intra vitam diagnosis of a patent E. multilocularis infection in final hosts is usually performed using flotation techniques, and copro-antigen ELISA methods have been employed, e.g. for epidemiological studies (Conraths and Deplazes, 2015; Liccioli et al., 2012). However, flotation methods have a low diagnostic sensitivity and E. multilocularis eggs are morphologically indistinguishable from the eggs of Taenia spp. (Liccioli et al., 2012). Thus for species identification of Taenia-type eggs, molecular techniques are needed (Knapp et al., 2014; Trachsel et al., 2007). Another possibility to diagnose E. multilocularis infection in a final host during the patent phase of infection is the extraction of DNA from faecal samples and subsequent amplification of E. multilocularis-specific DNA targets (Davidson et al., 2009; Isaksson et al., 2014; Knapp et al., 2014; Oines et al., 2014; Trachsel et al., 2007).<br />DNA extraction is a critical step for the detection of E. multilocularis genome fragments in faecal samples as the extraction process can be impaired by several factors, e. g. the kind of cell lysis (thermo-enzymatic or mechanical), or the quantity of the faecal sample to be processed. One further critical point of this step is to remove PCR copro-inhibitors during DNA extraction (Opel et al., 2010). In addition, the sensitivity may depend on the concentration of eggs and further sources of parasite DNA. Commercially available DNA extraction kits for faecal and/or environmental samples may be a good alternative for several published in-house methods for DNA extraction from E. mul<img src="http://www.RO4929097.COM/images/1-s2.0-S2093791114000699-gr2.jpg#" />tilocularis eggs (Davidson et al., 2009; Dyachenko et al., 2008; Mathis et al., 1996; Stefanic et al., 2004). The advantages of these kits are their standardized performance, easy handling and the speed of DNA extraction. A major disadvantage is that they are not adequate for sample quantities of more than 0.15–0.5g. DNA extraction kits for faecal and/or environmental samples from several suppliers have been applied in a number of studies in which DNA was extracted from eggs or oocysts of different parasites (Elwin et al., 2014; Herrmann et al., 2011; Knapp et al., 2014; Nonaka et al., 2009a; Nonaka et al., 2009b).<br />Comparisons of commercial kits  <img src="http://farm5.static.flickr.com/4318/35937681412_94ce43ba24.jpg" align="right" width="265" style="padding:10px;"/> used to extract DNA from different parasites or other microorganisms and matrices showed that it is essential to evaluate the performance of the kits before routine application (Elwin et al., 2014; Ferrand et al., 2014; Herrmann et al., 2011; Phillips et al., 2012; Scupham et al., 2007; van Tongeren et al., 2011). Herrmann et al. (2011) clearly demonstrated that only two out of six tested kits were suitable to extract DNA from Hammondia hammondi and Toxoplasma gondii oocysts in faecal samples.<br /><br><br />Materials and methods<br /><br><br />Results<br /><br><br />Discussion<br />DNA extraction is one of the most crucial steps in the molecular detection of E. multilocularis infections in the final hosts as well as in the species differentiation among Taenia spp. eggs. Little is known about the performance of such test kits, e.g. their analytical sensitivity, the removal of inhibitors present in faecal samples (copro-inhibitors) or limitations in DNA recovery. In previous studies (Rantakokko-Jalava and Jalava, 2002, 2011), it was shown that a careful validation of commercially available DNA extraction kits is crucial to select kits with optimal performance. Moreover, Herrmann et al. (2011) showed that the DNA isolation from oocysts of the protozoan parasites H. hammondi and T. gondii can be negatively influenced by selection of less appropriate DNA extraction kits. In the present study, we demonstrate that the performance of DNA extraction kits differs largely when used for isolating E. multilocularis-DNA from eggs of the parasite. To maintain the optimal performance of the kits, the amounts of faecal <a href='https://www.readmatter.com/'>matter</a>  used with each kit were those recommended by the manufacturers. They were therefore not exactly the same for all four kits (Table 1).
pitao 25, Jul 2017 toad0guitar (420 poena)
    

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