Detection of Mink (Mustela vison) DNA in Meat Products using Polymerase Chain Reaction PCR Assay
The study was to develop a Polymerase Chain Reaction (PCR) assay for specific detection of mink meat using designed pairs of primers based on mitochondrial D-loop. Mink meat is used as fraud ingredients of false mutton or dog meat in meat markets. This study was conducted to establish Polymerase Chain Reaction (PCR) method for the sensitive and specific detection of mink (Mustela vison) DNA in meat products. Six pairs of primers were designed from tandem repeat region of D-loop in mitochondria after alignment of the available sequences in the GenBank database. The specific pair of primers chosen from the six designed pairs by PCR generated specific fragments of 343 bp in length for mink. The specificity of detection was conducted with DNA samples of mink, blue fox, dog, raccoon dog, swine, sheep. Then amplification of positive reaction was observed only in mink species. In this study, no adverse effects of cooking and autoclaving were found on amplification of mink DNA fragments. Then the detection limit was found to be less than 1% in mixed meat products. The PCR method described in this study proved to be very sensitive and reliable in mink DNA identification. Thus, it could be considered as a further improvement method for the detection of mink DNA in meat products processed under different manufacturing conditions.
Received: June 15, 2012;
Accepted: September 14, 2012;
Published: November 19, 2012
Mink (Mustela vison) is a species of carnivore, Mustelidae family and
Mustela genus in taxonomy who originated from North America(Mustela
vision) and Europe Mustela lutreola. Minks as fur bearing animals
all over the world were precious descended from America Mustela vison.
(Niethammer et al., 1993; Wamberga
and Tausonb, 1998). Breeding and caring for fur bearing animals were conducted
originally in 1950s in China. Special economic animals have becoming the special
agriculture industry for its particular resources traits, economic values and
growing demand. Mink meat was mostly used as ingredients of animal feed and
fewly processed food. But, in some areas, mink meat was used as substitute of
other meat products because it is cheaper and easily available. Mink heart was
used for pharmaceutical industry especially due to cure the Rheumatic Heart
There are fraud phenomenons in meat and fur markets for various reasons such
as public health, religious factors and abnormal competition (Arslan
et al., 2006; Mane et al., 2006).
Because mink fur products and other appendix are expensive and limited, merchants
usually substitute them with other products to obtain lots of economic profits.
Such as mink fur was substitution for low priced animal fur even the artificial
fur. The mink hearts for drugs are substitution for chicken hearts or rabbit
hearts. In recent years, the mutton adulterations have becoming growing increased
substitution for mink meat, fox meat or raccoon dog meat (Sun
et al., 2006). At present, the quality control of meat products mostly
depends upon traditional methods which mainly include macroscopical identification,
microscopical examination and physical and chemical experiment. It is difficult
to identify the fraud using the traditional methods especially with the development
of processing technologies (Girish et al., 2005).
So, it is very necessary to identify mink DNA in order to inhibit the fraud
phenomenon. Food safety has aroused highly attention of consumers and governments
all over the world especially the meat products, animal feed.
The composition of mtDNA has no complicated intron, pseudogene or repetitive
sequence which is simpler in complexity than nuclear DNA (nDNA) (Gray,
1989). Analysis of mitochondrial DNA (mtDNA) sequences has gained particular
attention these years. The mtDNA is of maternal inheritance and has no recombination
in all vertebrates, so that the sequence of mtDNA is more conservative (Rokas
et al., 2003). However, the rate of base substitution on mtDNA is
higher than that of nDNA, causing a rapid evolution (Stoneking
and Soodyall, 1996). On average, there are about 800-1000 mitochondria per
cell and each mitochondrion contains 2-6 circular DNA molecules, making mtDNA
a naturally amplified source of genetic variation (Girish
et al., 2004).
DNA based assays are gaining popularity in meat species identification due
to their stability at high temperature and conserved structure within all individual
of the species (Calvo et al., 2002; Girish
et al., 2004). The PCR assays are employed for identification of
species origin of meat using random primers (Saez et
al., 2004). Demmel et al. (2008) has
reported the method of detection of lupine (Lupinus spp.) DNA in processed
foods using real-time PCR. Many researchers (Fajardo et
al., 2007; Tang et al., 2002a, b)
identified different animals of Cervidae by using species-specific PCR method.
Simultaneous detection of pathogenic vibrio species using multiplex real-time
PCR has been reported by Kim et al. (2012). PCR-restriction
fragment length polymorphism (PCR-RFLP) has been applied for the identification
of deer-derived ingredients in the deer products (Kim et
al., 2001; Matsunaga et al., 1998). The
method of multiplex PCR to detect animal ingredients in feedstuffs or food products
has been developed by most researchers recently (Lin and
Hwang, 2008; Mane et al., 2009; Dalmasso
et al., 2004).
This study was aimed to develop a PCR method to rapid detection the mink DNA
in meat products especially in mutton and dog products, which provides a more
precise detection of mink species origin for complex meat samples.
MATERIALS AND METHODS
Meat and blood samples: The study was performed during 1st November,
2011 to 20th December, 2011. Blood samples were collected in the first two weeks
during this period. Whole blood samples (1 mL) of mink, blue fox, dog, raccoon
dog, swine, sheep were obtained in fur animal breeding base or animal slaughterhouse
in Jilin province. The samples were collected in tube with Ethylene Diamine
Tetra Acetate (EDTA) as anticoagulant. The collected blood samples were preserved
at-20°C till DNA isolations.04.
In this study, the analysed meat samples were collected from local slaughterhouses which were mink, blue fox, silver fox, raccoon dog, pork, mutton and dog meat. Refrigerated packed mutton and dog meat were bought from a local store in Jilin province (China) and some meat products were bought from chaffy dish restaurant. These products were claimed from mutton or dog meat as described in their labels. Samples were kept at -20°C till further processing.
DNA extraction and purification: Genomic DNA was extracted from each blood sample according to manufacturer of Genomic DNA Extract Kit (TianGen China). Each sample of meat products was weighed 200 mg. Genomic DNA was extracted from all samples using standard procedures with minor modifications. The lysis buffer was modified with 0.5% SDS, 0.5% Triton X-100, 10 mM Tris-Cl pH 7.6, 10 mM Na2EDTA, 8 mM MgCl2 and 8 mM NaCl. The quality of genomic DNA was checked by horizontal submarine agarose gel electrophoresis using 1.0% agarose. The concentration of DNA was estimated by spectrophotometry (SPECORD S600, Analytik Jena AG, Jena, Germany) and the quality and purity of DNA was evaluated by A260/A280.
Subsequently, the DNA extracts were purified with the QIAquick PCR purification kit (TianGen China) according to the manufacturer. DNA samples were diluted to 10, 5, 1, 0.5 and 0.1%, respectively in order to evaluate the test sensitivity later.
Primers design: D-loop region sequences and complete mitochondrial genome were obtained from available sequences in National Center for Biotechnology Information (NCBI) GenBank which contained 39 of Mustelidae family. Alignment of complete mitochondrial genome from Martes zibellina (NC011579), Gulo gulo (AM711901), Nyctereutes procyonoides (GU256221), Vulpes vulpes (Q374180), Cervus nippon hortulorum (U457433), Canis lupus familiaris (NC002008), Canis lupus chanco (NC010340) and Oryctolagus cuniculus (NC001913) was performed using MEGA5.0. Specific primers for mink species were then designed based upon D-loop region sequence of Martes zibellina (NC011579) using primer designing soft-ware called Primer 5.0. Then earlier 6 pairs of primer were designed to be chosen, each of which consisted of a forward primer(F) and a reverse primer (R). Designing primers was based on the principle which was base composition and annealing temperature of primers were consistent with each other as far as possible and each pair of primers had intraspecies-universality and interspecies-specificity. The designed primers were synthesised by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd., Shanghai, China.
Specific primer selection and the PCR conditions: In a preliminary phase
of this research, the selection of the specific primers among the 6 pairs was
assessed and chosen with DNA extracted from whole blood samples in minks (Fig.
1). The reaction mixture was prepared in a 500 μL PCR tube (AXYGEN,
USA) in a total volume of 25 μL containing 5 μL of forward and 5 μL
of reverse primer, 12.5 μL of PCR Mix (containing buffer, dNTP, Taq
DNA polymerase) (TaKaRa Biotechnology Co. Ltd., Dalian, China), 8.5 μL
of ddH2O, 2 μL of DNA template. The most specific primer called
primer1 was as follows:
|| 5'CTTCAACCTCAACATCATCACC 3
||5' GACATACATTGTATTCATTCTAAGCG 3'
||PCR amplification of mink mitochondrial DNA genome extracted
from blood samples with the 6 pairs of primers separately, Lane M: D2000
bp marker, Lane 1-6: Mink template with primer1-6
|| PCR conditions parameters
The PCR was programmed on 2720 thermal cycler (Applied Biosystems, USA) and the PCR cycling conditions parameters are given in Table 1.
Amplified product detection: The specific primer was used for amplification in the species of mink, blue fox, silver fox, raccoon dog, pork, mutton and dog (Fig. 2). 0.4 g of agarose (TakaRa, China) was put in 40 mL of 1x TBE solution (Fermentas, USA) and heated to completely dissolve the agarose. Then 1 drop of (approximately 5%) ethidium bromide solution was added as gel visualising agent and mixed thoroughly. The PCR product was finally analysed using UV transilluminator and documented by gel documentation system (Alpha Imager, USA). The ready to use 100 bp ladders (Fermentas, USA) was used for present work. PCR amplified products were analyzed by electrophoresis on 1% agarose gel (TakaRa, China) contained Ethidium Bromide run in TBE buffer for 90 min at 80 V.
Sequencing and alignment: Each amplified fragment was purified by PCR
Products Purification Kit (Spin-column) (TaKaRa Biotechnology Co. Ltd., Dalian,
China) and cycle was sequenced (both strands) using PCR derived primers.
||PCR amplification of selected specific primers with DNA genome
samples, Lane M: D2000 bp marker, Lane 1: Mink, Lane 2: Blue fox, Lane 3:
Silver fox, Lane 4: Raccoon dog, Lane 5: Pork, Lane 6: Mutton, Lane 7: Dog,
Lane 8: Negative control
The dideoxy chain termination method was performed by Shanghai Sangon Biological
Engineering Technology and Services Co. Ltd., Shanghai, China. The nucleotide
sequences were aligned with sequence of Martes zibellina (NC011579) downloaded
from GenBank database. Similarity of amplified segment and template was 99%.
The amplified segment sequence was as follows:
Detection limit (PCR sensitivity): The specificity of PCR assay was
tested with DNA of other animal species used in this study. The DNA templates
were diluted with ddH2O containing 100%, 50,10, 5, 1, 0.5 and 0.1%
mink DNA in order to test the sensitivity of PCR reaction. The non-targeted
species were mink, blue fox, silver fox, raccoon dog, pork, mutton and dog meat.
Finally, the detection limit was 0.5% level of adulteration of mink DNA in admixed
meat products (Fig. 3). The detection limit quantity was 0.05
ng for mink.
The aim of the study was to develop and evaluate a method for detection of
mink species in meat and meat products even processed under different manufacturing
conditions. The variable regions of the mitochondrial gene are present in thousands
of copies per cell (Greenwood and Paabo, 1999) which
increases the probability of achieving a positive result due to processing conditions
(Bellagamba et al., 2003). The species-specific
PCR assay as a low-cost, precise and rapid testing method is indispensable to
avoid unfair market competition and protection of consumer from fraudulent practices
of meat adulteration. Some workers had suggested that mitochondrial markers
were more efficient than nuclear markers for the purpose of detection and authentication
animal species (Hopwood et al., 1999).
||PCR amplification of mink mitochondrial D-loop gene with various
dilution levels up to 0.1%, Lane M: D2000 bp marker, Lane 1: 100% level,
Lane 2: 50% level, Lane 3: 10% level, Lane 4: 1% level, Lane 5: 0.5% level,
Lane 6: 0.1% level, Lane 7: Negative control
The mitochondrial DNA was targeted to design species-specific primers, because
mitochondrial DNA is maternally inherited so normally only one allele exists
in an individual and thus no sequence ambiguities are to be expected from the
presence of more than one allele (Unseld et al.,
1995). The specific pair of primers was designed based on mitochondrial
D-loop for amplification of 343 bp DNA fragments from mink DNA. Earlier, Calvo
et al. (2002) also successfully developed swine-specific primers
for detection of pork in wide range of meat and meat products in raw and cooked
meats, sausages, cured meat products, hamburgers and pates.
The species-specific PCR assay was found to be precise, sensitive and rapid methods for identification of species which can be used for routine analysis of meat species, even in admixed meat and meat products under different processing conditions. Thus, it can be concluded that it was a potentially reliable technique and useful tool for detection of mink meat from other animals to protect the consumers from fraudulent practices of meat substitution.
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