Food Forensics Analysis of Food , Raw and Processed Materials With Molecular Biological Methods
-
Upload
alina-ioana -
Category
Documents
-
view
19 -
download
1
Transcript of Food Forensics Analysis of Food , Raw and Processed Materials With Molecular Biological Methods
Research article
Food forensics: Analysis of food, raw and processed materials with
molecular biological methods
R. Schubbert *, W. Hell, T. Brendel, S. Rittler, S. Schneider, K. Klopper
Eurofins 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 identification 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 identification 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
quantification of fractions different species contributed to a sample.
# 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Species identification; Plant variety identification; Processed food and non-food products; Trace material; Forensic application
www.elsevier.com/locate/FSIGSS
Available online at www.sciencedirect.com
Forensic Science International: Genetics Supplement Series 1 (2008) 616–619
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 difficult 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-specific discrimination power and preferably
short amplicons. The identification of species from material
lacking cells (e.g. animal fibres) makes the use of genomic
markers often impossible, hence suitable mitochondrial markers
have to be used [1,2]. We present test methods for species
identification from animal fibres, processed food and plants as
well as plant variety identification and quantification methods.
* Corresponding author at: Eurofins Medigenomix, Applied Genetics, Fraun-
hofer Str. 22, 82152 Planegg/Martinsried, Germany. Tel.: +49 89 89 98 92 22;
fax: +49 89 89 98 92 92.
E-mail address: [email protected] (R. Schubbert).
1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2008.01.003
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 identification from animal fibres and
quantification
Species detection was done using mtDNA. DNA was
extracted from yarn, draperies and raw material. Specific
primers were developed for qualitative and quantitative
detection of sheep, goat, yak and camel DNA by RT-PCR.
2.2. Species identification 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.
Fig. 1. RT-PCR result of a wool sample (a) and a yarn sample (b).
R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616–619 617
2.3. Plant species identification from dried plant material
PCR products of ITS 1 of nuclear rRNA gene [4] were
generated from DNA extracted from dried ginseng slices
Fig. 2. Sequence alignment of the h3 gene of M. edulis and M
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.
. chilensis; sequence differences are marked with arrows.
R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616–619618
2.4. Plant variety identification
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
We demonstrate successful species and variety determina-
tion in various processed materials, including quantification of
ingredients in blends and mixtures.
3.1. Species identification from animal fibres and
quantification
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
fibres from two species the ratio of species fibres can be
Fig. 3. Results of the Blast search for the query sequence of genus Pan
determined by comparison of Ct ratios from reference samples
and test sample (green arrow).
3.2. Species identification 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 identification of M. chilensis (Fig. 2a) and M.
edulis possible (Fig. 2b).
3.3. Plant species identification from dried plant material
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 identified as Panax ginseng.
3.4. Plant variety identification
By comparison of allelic patterns of 10 different markers
with known allele patterns of Basmati rice varieties, doubtful
samples could be identified as non-Basmati rice (not shown),
ax identifying the query sequence as Panax ginseng (see also text).
Fig. 4. Basmati rice microsatellite profiles of different rice varieties (see also text).
R. Schubbert et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 616–619 619
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.
Conflict of interest
None.
References
[1] I. Pfeiffer, et al., Diagnostic polymorphisms in the mitochondrial cyto-
chrome b gene allow discrimination between cattle, sheep, goat, roe buck
and deer by PCR–RFLP, 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) 840–847.
[3] J.M. Eirin-Lopez, et al., Molecular evolutionary characterization of the
mussel Mytilus histone multigene family: first record of a tandemly
repeated unit of five histone genes containing an H1 subtype with ‘‘orphon’’
features, J. Mol. Evol. 58 (2) (2004) 131–144.
[4] G.M. Plunkett, et al., The classification 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.