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

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