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Forensic Science International: Genetics Supplement Series 1 (2008) 616619

Research article

Food forensics: Analysis of food, raw and processed materials with molecular biological methods
pper R. Schubbert *, W. Hell, T. Brendel, S. Rittler, S. Schneider, K. Klo
Eurons Medigenomix, Fraunhofer Str. 22, 82152 Planegg/Martinsried, Germany Received 17 August 2007; received in revised form 18 January 2008; accepted 23 January 2008

Abstract The identication of species is vital for product quality control as well as for detection of fraud and even more so for the investigation of crimes when biological trace material is found. They all have in common that the correct assignment of biological samples to species is aggravated due to highly degraded (e.g. due to heavy processing) or low amounts of DNA. Here we present examples of successful identication of species and even varieties in raw and processed materials such as textiles, seafood and plant products. We also show the use of two different methods for quantication of fractions different species contributed to a sample. # 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Species identication; Plant variety identication; Processed food and non-food products; Trace material; Forensic application

1. Introduction In the food, non-food and pharmaceutical production chain, premium products are often blended with low-quality products for several reasons. The detection of these manipulations is often difcult because of low amounts or strongly degraded DNA (due to processing). For our routine work for our customers we optimized and developed new methods. The methods are transferable to forensic casework, e.g. in cases of fraud or when trace material is found on a suspect and has to be compared with traces found on the scene of crime. Standard methods are rarely suitable for such investigations as they either require high amounts of DNA or known sequence information. New genes and methods have to be investigated with a high inter-specic discrimination power and preferably short amplicons. The identication of species from material lacking cells (e.g. animal bres) makes the use of genomic markers often impossible, hence suitable mitochondrial markers have to be used [1,2]. We present test methods for species identication from animal bres, processed food and plants as well as plant variety identication and quantication methods.

2. Materials and methods DNA was extracted from various sources using different commercial DNA extraction kits from Macherey & Nagel. Qualitative and quantitative analyses were performed both with genomic and mitochondrial DNA with various techniques such as microsatellite analysis, sequencing analysis and real-time PCR (RT-PCR). 2.1. Species identication from animal bres and quantication Species detection was done using mtDNA. DNA was extracted from yarn, draperies and raw material. Specic primers were developed for qualitative and quantitative detection of sheep, goat, yak and camel DNA by RT-PCR. 2.2. Species identication from processed food DNA was extracted from mussel tissue with different Macherey & Nagel kits depending on the freshness of mussels. At the time of analysis, no sequence information for mitochondrial genes was available for the species. Primers were designed for several histone genes [3] and the PCR products were sequenced using the ABI Big Dye Terminator 3.1 kit.

* Corresponding author at: Eurons Medigenomix, Applied Genetics, Fraunhofer Str. 22, 82152 Planegg/Martinsried, Germany. Tel.: +49 89 89 98 92 22; fax: +49 89 89 98 92 92. E-mail address: (R. Schubbert). 1875-1768/$ see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2008.01.003

R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616619


Fig. 1. RT-PCR result of a wool sample (a) and a yarn sample (b).

2.3. Plant species identication from dried plant material PCR products of ITS 1 of nuclear rRNA gene [4] were generated from DNA extracted from dried ginseng slices

declared as Panax ginseng. Primers were designed from published sequences [4]. The PCR products were sequenced and compared to Panax sequences published in Genbank using BLAST search.

Fig. 2. Sequence alignment of the h3 gene of M. edulis and M. chilensis; sequence differences are marked with arrows.


R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616619

2.4. Plant variety identication A set of established microsatellite markers [5] was applied to categorise rice samples of various processing stages to differentiate and identify the varieties of Basmati rice (Oryza sativa). The method is also applied to quantify the proportions of rice varieties within a sample as blends containing up to seven percent non-Basmati still qualify as real Basmati rice. 3. Results

determined by comparison of Ct ratios from reference samples and test sample (green arrow). 3.2. Species identication from processed food The two mussel species differed at two positions (marked by red and green arrows) in their histone H3 gene sequences making a reliable identication of M. chilensis (Fig. 2a) and M. edulis possible (Fig. 2b). 3.3. Plant species identication from dried plant material

We demonstrate successful species and variety determination in various processed materials, including quantication of ingredients in blends and mixtures. 3.1. Species identication from animal bres and quantication Data presented show results of the analysis of two wool samples. One sample of raw wool (Fig. 1a) contains wool from sheep (red arrow, FAM) and goat (cashmere) (blue arrow, VIC), another yarn sample (Fig. 1b) only sheep. In the VIC channel only background signal is detectable. In samples with animal bres from two species the ratio of species bres can be

BLAST search and comparison of 108 bp section of the PCR product showed no differences with the published Panax ginseng sequence (Fig. 3a), one difference with Panax quinqefolius (Fig. 3b), two with Panax japonicus (Fig. 3c) and three with Panax stipuleanatus (Fig. 3d). The ginseng slices could thus be identied as Panax ginseng. 3.4. Plant variety identication By comparison of allelic patterns of 10 different markers with known allele patterns of Basmati rice varieties, doubtful samples could be identied as non-Basmati rice (not shown),

Fig. 3. Results of the Blast search for the query sequence of genus Panax identifying the query sequence as Panax ginseng (see also text).

R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616619


Fig. 4. Basmati rice microsatellite proles of different rice varieties (see also text).

mixtures of different Basmati-varieties and/or non-Basmati rice or as unadulterated Basmati (Fig. 4). Adulteration of products with non-Basmati rice even in low concentrations can be detected. The method is suited for the analysis of brown rice, white, par-boiled and even highly processed rice. The above examples show, traditional marker systems and methods may not always be available or suitable for correct species assignment of biological material. The described approaches could be expanded to other species where primer/ marker information is sparse or unavailable. This will aid to determine the plant or animal origin of biological material in forensic casework where other methods fail. Conict of interest None.

[1] I. Pfeiffer, et al., Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCRRFLP, BMC Genet. 5 (2004) 30. [2] D.R. Foran, et al., DNA-based analysis of hair to identify species and individuals for population research and monitoring, Wildl. Soc. Bull. 25 (4) (1997) 840847. [3] J.M. Eirin-Lopez, et al., Molecular evolutionary characterization of the mussel Mytilus histone multigene family: rst record of a tandemly repeated unit of ve histone genes containing an H1 subtype with orphon features, J. Mol. Evol. 58 (2) (2004) 131144. [4] G.M. Plunkett, et al., The classication of Araliaceae: testing traditional systems using insights from nuclear (ITS) and plastid (trnL-trnF) sequence data, NCBI Database PopSet. [5] Code of Practice on Basmati Rice (COP), British Retail Consortium, British Rice Millers Association, The Rice Association, July 2005; Survey on Basmati Rice, Food Standard Agency (FSA), UK, March 2004.