Identification of Horse Meat and Beef using a Polymerase Chain Reaction Based Method with Cytochrome b Gene
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Date
2013
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Journal ISSN
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Publisher
Uva Wellassa University of Sri Lanka
Abstract
Adulteration of meat and meat products are taking place in many parts of the world and it is
believed that numerous such incidents have even occurred in Sri Lanka as well. Demanding
meat and meat products are being adulterated with cheaper or unconventional meat types (eg.
dog, horse or rat meat) with phenotypic similarities. This situation created a scenario where the
food analyst from many developing countries needs to pay special attention to identify meat. A
recent disclosure in UK about an adulteration of beef with equine meat created a paranoid
situation, which drastically affected genuine brands produced by large companies and
international food chains (Thomson, 2013). Hence, the adulteration must be prevented to ensure
traceability of meat from farm to form. In general, molecular methods facilitate accurate and
more reliable analysis of meat adulteration. Compared to nuclear DNA, application of
mitochondrial DNA (mtDNA) in meat identification experiments provides many advantages,
such as maternal inheritance and ubiquitous natures (Kvist, 2000). Thus, mtDNA can be used
when the evidentiary material supply is limited. Among the Mitochondrial genes mitochondrial
cytochrome b (Cyt b) gene has often used to detect the source of meat (Farias, et al., 2001).
Hence, the aim of this study is to establish qualitative polymerase chain reaction method to
detect horse meat adulteration in beef using mitochondrial DNA Cyt b region.
Methodology
Beef and horse (Equus ferus caballus) meat samples were collected from slaughter house at
Dematagoda and from Faculty of Veterinary Medicine and Animal Science, at University of
Peradeniya, respectively. Genomic DNA was extracted following the protocol as described in
Abdel-Rahman et al. (2009) with modifications. First, 600 mg of tissue was homogenized in
6000 μL STE buffer and 48 μL of 10% SDS and 24 μL of proteinase K (10 mg/mL) was added.
Then, the mixture was incubated at 37 °C overnight. After that, DNA was purified by equal
and chloroform–isoamylalcohol
(24:1), successively. Then, DNA was precipitated by adding chilled ethanol in the presence of 3
M sodium acetate. The resulting pellet was washed with 70% ethanol, air-dried and
subsequently dissolved in 80 μL of TE buffer.
Species specific primers were designed (horse forward (HF) - ATC ATC ACA GCC CTG GTA
GTC GTA CAT, horse reverse (HR) - ATG TGG AGG GTG GGG ATG AGT GCT A, cattle
forward (CF) - CAT CGG CAC AAA TTT AGT CG and cattle reverse (CR) - GAG CTA GAA
TTA GTA AGA GGG CC) to amplify mitochondrial Cyt b gene of cattle and horse. The PCR
products were electrophoresed on 2% agarose gel containing 0.5 µg/mL Ethidium bromide and
were visualized and imaged using a UV trans-illuminator (Gel Dox XP+ system, BioRad) and
gel documentation system (Image Lab 3.0, BioRad) to distinguish the species origin. Furthermore, to investigate the detection limit of the PCR system, DNA was extracted from 600
mg of beef which was mixed separately with 1%, 5% and 20% of horse meat.
Results and Discussion
MtDNA evolve at a much faster rate than nuclear DNA. At the same time different regions of
the mitochondrial genome would evolve at different rates. Therefore, mtDNA maintain variable
regions but with a certain level of conservation. Similarly, mitochondrial Cyt b gene contains
both slow and rapid evolving regions with conservative and variable regions. The evolution of
the Cyt b gene is prevented due to the functional restrictions in the conservative regions (Farias,
et al., 2001). Therefore, Cyt b gene is used to identify horse meat from beef as the sequence
variability in Cyt b gene between different species is extremely high.
Description
Keywords
Animal Sciences, Meat, Meat Production, Food Science, Food Technology