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. 2014 Jul 17:4:5730.
doi: 10.1038/srep05730.

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)

M C Lecrenier  1 , Q Ledoux  2 , G Berben  3 , O Fumière  3 , C Saegerman  4 , V Baeten  3 , P Veys  3
Affiliations

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)

M C Lecrenier et al. Sci Rep. .

Abstract

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

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Figures

Figure 1
Figure 1. Micrographs of processed bones hybridized using an ATTO 565-labelled bovine DNA probe.
(a–c): Bone particle in brightfield of bovine (a), porcine (b) and ashed bovine origin (c). (d–f): Corresponding images in epifluorescence for bovine (d), porcine (e) and ashed bovine (f) bone. Composite images of the brightfield and epifluorescence images shown for bovine (g), porcine (h) and ashed bovine (i) bone. The arrows point to in situ hybridization spots. Scale bars = 20 μm.
Figure 2
Figure 2. Micrographs of bovine processed bone hybridized using an ATTO 565-labelled bovine DNA probe.
Bovine bone particle in brightfield (a) and its corresponding image in epifluorescence (b). (c) shows the composite image of the brightfield and epifluorescence images. The arrows point to an internal structure within the lacuna, which corresponds to the hybridization spot. Scale bars = 10 μm.
See this image and copyright information in PMC

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