Food Additives & Contaminants: Part A
ISSN: 1944-0049 (Print) 1944-0057 (Online) Journal homepage: http://www.tandfonline.com/loi/tfac20
A lab-on-a-chip-based multiplex platform to detect
potential fraud of introducing pig, dog, cat, rat and
monkey meat into the food chain
Md. Abdur Razzak, Sharifah Bee Abd Hamid & Md. Eaqub Ali
To cite this article: Md. Abdur Razzak, Sharifah Bee Abd Hamid & Md. Eaqub Ali (2015): A
lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig, dog,
cat, rat and monkey meat into the food chain, Food Additives & Contaminants: Part A, DOI:
10.1080/19440049.2015.1087060
To link to this article: http://dx.doi.org/10.1080/19440049.2015.1087060
Accepted author version posted online: 05
Oct 2015.
Published online: 08 Oct 2015.
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Date: 19 October 2015, At: 19:41
Food Additives & Contaminants: Part A, 2015
http://dx.doi.org/10.1080/19440049.2015.1087060
A lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig, dog, cat,
rat and monkey meat into the food chain
Md. Abdur Razzaka, Sharifah Bee Abd Hamida and Md. Eaqub Alia,b*
a
Nanotechnology and Catalysis Research Centre (NanoCat), University of Malaya, Kuala Lumpur 50603, Malaysia; bCentre for
Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur 50603, Malaysia
Downloaded by [University of Malaya] at 19:41 19 October 2015
(Received 15 July 2015; accepted 23 August 2015)
Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat,
rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers
need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need,
here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of
five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of
mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species
under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used
in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA.
The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of
meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect
0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only
one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species
in a single assay platform.
Keywords: lab-on-a-chip-based multiplex PCR; haram meat-species; extreme sterilisation; commercial food matrices
Introduction
The world is getting increasingly busier, and a growing
proportion of the population faces time-constraints in
making their own meals. Consequently, they are forced
to accept whatever they can have from a nearby restaurant or grocery store (Ali et al. 2013; Rahman et al.
2014). Thus, the demands and prospects of restaurant
businesses and ready-made foods, such as burgers, pizzas, hot dogs, sandwiches, soups, cookies, candies and
creams, are growing fast (Van Der Spiegel et al. 2012).
However, labelling does not necessarily always guarantee total control “from farm-to-fork”. One of the
obvious regions might be the massive and ongoing
innovations in processing and packaging technologies
(McMillin 2008; Cammà et al. 2012). Furthermore, the
recent scandals of horse meat in Europe and pig and rat
meats in China (Ali et al. 2014; Premanandh 2013) and
the repeated addition of prohibited food items such as
pork, dog and cat, meats with various dishes have
alarmed consumers into demanding testing for the presence of prohibited ingredients in marketed foods
(Mohamad et al. 2013; Karabasanavar et al. 2014;
Kitpipit et al. 2014; Rashid et al. 2015). As a result,
the European Commission has emphasised food safety
as a key priority in food policy and health (Regulation
*Corresponding author. Email: [email protected]
© 2015 Taylor & Francis
EC No. 178/2002). Some other countries, such as
Malaysia, Indonesia, China, Thailand, Singapore and
Brazil, have established trustworthy certification bodies
to control and protect public health, religious credence,
fair-trade business and wildlife linked to food falsification and/or mislabelling, especially of animal origin
(Rohman et al. 2011; Rahman et al. 2015).
The enforcement of labelling regulations requires
sensitive, reliable and easily performable scientific
methods to verify trace ingredients in processed and
unprocessed foods (Ghovvati et al. 2009; Fajardo et al.
2010; Doosti et al. 2011; Herrero et al. 2011;
Cawthorn et al. 2013; Kesmen et al. 2013; Hou et al.
2014; Karabasanavar et al. 2014). Although lipid- and
protein-based methods can be applied for the biomarker-based molecular authentication schemes (Buckley
et al. 2010; Sentandreu & Sentandreu 2011; Von
Bargen et al. 2013), these techniques are laborious,
cumbersome and need expensive instruments and
skilled manpower for operation and complicated statistical analysis to draw a conclusion (Che Man et al.
2005; Nurjuliana et al. 2011; Van Der Spiegel et al.
2012). Moreover, protein-and lipid-based methods are
less reliable because both the amount and type of fats
and fatty acids could be modified during the processing
treatments (Rohman et al. 2011). In contrast, Codon
Downloaded by [University of Malaya] at 19:41 19 October 2015
2
Md. A. Razzak et al.
degeneracy, superior stability, and universal traceability of DNA in all cells as well as lower-cost instrumentation have made DNA based methods feasible for
practical analysis (Kitpipit et al. 2014; Hou et al. 2014;
Safdar & Junejo 2015) and hence a myriad of DNAbased assays including species-specific polymerase
chain reaction (PCR; Karabasanavar et al. 2014;
Rahman et al. 2014), PCR-RFLP (Dooley et al. 2005;
Chen et al. 2014; Rashid et al. 2015), PCR product
sequencing (Ali et al. 2013), real-time PCR (Kesmen
et al. 2013) and DNA barcoding (Di Pinto et al. 2013;
Lamendin et al. 2015) have been documented for meat
species authentication. Species-specific PCR seems to
be the best because it is often conclusive and low-cost
in comparison to other methods such as single nucleotide polymorphism (SNP) analysis, PCR-RFLP, PCRRAPD and DNA barcoding (Ballin 2010; Bottero &
Dalmasso 2011; Ali et al. 2014; Karabasanavar et al.
2014). Multiplex PCR assays with species-specific primers are very promising because they offer multiple
target detection in a single assay platform, reducing
both cost and time (Matsunaga et al. 1999; Zha et al.
2010, 2011; Safdar et al. 2014). Although several
multiplex PCR assays have been documented for the
identification of various animal species (Matsunaga
et al. 1999; Dalmasso et al. 2004; Di Pinto et al.
2005; Zhang 2013; Kitpipit et al. 2014), none of
them have aimed at the authentication of five prohibited species in religious food, such as halal food. Here
we report the development and detailed verification of
a multiplex PCR assay for the detection of pig, dog,
cat, rat and monkey DNA under various commercial
food matrices such as beef and chicken meatballs,
burgers and frankfurters, which are popularly consumed across the world.
Methods and materials
Meat samples collection
Dog (Canis lupus familiaris), cat (Felis catus) and rat
(Rattus rattus) meats were collected in triplicates from
Dewan Bandaraya Kuala Lumpur, Air Panas, Kuala
Lumpur, Malaysia. Monkey (Macaca fascicularis)
meats from three different monkeys were obtained
from Wildlife Malaysia, Cheras, Kuala Lumpur. Pork
was purchased in triplicates from three different vendors from Chinese wet market in Seri Kambangan,
Selangor, Malaysia. The most commonly used commercial meat (beef (Bos taurus), chicken (Gallus gallus), goat (Capra hiscus), lamb (Ovis aries), buffalo
(Bubalus bubalis), venison (Odocoileus virginianus),
duck (Anas platyrhychos), pigeon (Columba livia)
and quail (Coturnix coturnix)), expensive fish species
(salmon (Salmo salar), cod (Gadus morhua), tuna
(Thunnus orientalis), carp (Cyprinus carpio), rohu
(Labeo rohita) and tilapia (Oreochromis niloticus))
and five different halal-branded (each of beef and
chicken) meatballs, seven burgers, and eight frankfurter items were purchased in triplicates on three different days from the various wet and supermarkets across
Malaysia. All meat samples and products were transported under ice-chilled condition (4°C) and stored
frozen at −20°C until use to prevent natural and enzymatic decompositions of meats and DNAs.
Preparation of dummy meat products
The most popularly consumed ready-to-eat meat products – namely, meatball, burger and frankfurter of
both beef and chicken – were prepared to validate the
reliability of the developed assay. To simulate commercial meat products, meatballs, burgers and frankfurters were prepared following Ali, Hashim, Mustafa,
et al. (2012), Ali et al. (2013) and Rahman et al.
(2014), respectively (Table 1). Prepared ready-to-eat
meat products were subjected to autoclave at 121°C
under 45 psi pressure for 2.5 h to simulate extensive
cooking and boiling effects (Rahman et al. 2014,
2015). All prepared samples were stored at −20°C
prior to DNA extraction.
Table 1.
Formulation of ready-to-eat model meat products (g).
Meatball
(≥35 g/piece)
Ingredients
Burger
(≥85 g/piece)
Frankfurter
(≥70 g/piece)
Beef Chicken Beef Chicken Beef Chicken
Minced meat 23 a
Soy protein
3
Starch/
5
breadcrumb
Chopped
1
onionb
Chopped
0.1
gingerb
0.75
Cumin
powderb
Garlic powderb 0.5
Black pepperb 0.14
Tomato paste
1.5
Butter
1.5
Egg
Salt
SA
SA
Othersc
23 a
3
5
60a
10.8
8
60a
10.8
8
45a
7.5
6.5
45 a
7.5
6.5
1
3
3
2.5
2.5
0.1
0.2
0.2
0.15
0.15
0.75
1
1
0.75
0.75
0.5
0.14
1.5
1.5
0.75
0.25
2.5
2.5
1
SA
SA
0.75
0.25
2.5
2.5
1
SA
SA
0.5
0.23
2
2.5
0.5
0.15
2
2.5
SA
SA
SA
SA
SA
SA
Notes: a1%, 0.5%, and 0.1% of all target meats (pig, dog, cat, monkey
and rat) were added both individually and collectively with a balanced
amount of minced chicken and beef to make ≥35 g, 85 g, and 70 g
specimen of each meatball, burger, and frankfurter, respectively.
b
Amounts are in approximate values and some items were taken in
teaspoon measurements.
c
Enhancers and flavouring agents.
SA, suitable amounts.
Food Additives & Contaminants: Part A
Downloaded by [University of Malaya] at 19:41 19 October 2015
DNA extraction from raw meats and meat products
Total DNA of all meat and fish samples were extracted
using Yeastern Genomic DNA Mini Kit (Yeastern Biotech
Co., Ltd, Taipei, Taiwan) from 20 mg of muscle tissues
following manufacturer’s instruction. DNA from model
and commercial meat products was extracted from
100 mg specimen. First, the sample was grinded with a
micropestle in a 1.5-ml micro-centrifuge tube to make a
pulp followed by the addition of 20 µl of Proteinase K.
The mixture incubated at 60°C for 30 min to lyse the
sample. After adding 400 µl of lysis buffer, the sample
mixture was incubated again at 60°C for 20 min to ensure
the clarity of sample lysate. Subsequent steps followed the
instructions given by the kit manufacturer (Yeastern
Biotech Co., Ltd, Taipei, Taiwan). DNA from plant species (wheat (Triticum aestivum), tomato (Solanum lycopersicum), garlic (Allium sativum), onion (A. cepa) and
pepper (Capsicum annuum)) was extracted using the
CTAB method according to Rahman et al. (2014).
Concentration and purity of the extracted DNA were
determined by UV-Vis Spectrophotometer (Libra S80,
Biochrom Ltd, Cambridge, UK).
3
species-specific primers targeting ND5 gene for pig and
monkey, ATPase 6 for dog and rat, and cytochrome b
for cat species. The whole genome sequences of pig
(AF034253.1), monkey (FJ906803.1), dog (NC_002008.4),
rat (NC_012374.1) and cat (NC_001700.1) were retrieved
from the NCBI database (http://www.ncbi.nlm.nih.gov/) and
were aligned using the ClustalW sequence alignment tool
(Thompson et al. 1994) to select the inter-species hypervariable and intra-species conserved regions. The regions
of the noted gene sites so found were used to design species-specific primers for pig, dog, rat, monkey and cat
(Table 2). The further checking of mismatches to all other
species either at the 3′ position or, where possible, for both
forward and reverse primers was performed by MEGA5
software (Tamura et al. 2011). The designed primers were
also screened for unique specificity to eliminate cross-species binding with other animal or plant species using the
online BLAST local alignment tool in the NCBI database
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). All designed primers were purchased from IDT, USA.
Simplex PCR optimisation
Biomarker selection
Species-specific primers were designed by targeting mitochondrial genes because they are well protected by mitochondrial membrane, maternally inherited and present in
multiple copies per cell (Xin et al. 2006). Among the
mitochondrial genes, NADH dehydrogenase subunit 5
(ND5) and ATPase subunit 6 offer appropriate target length,
sufficient degree of intra-species conserved regions and
interspecies polymorphism, and available sequence database for most animals and plants (Da Fonseca et al. 2008;
Kitpipit et al. 2014). On the other hand, moderate evolutionary rate and clear patterns have made cytochrome b
(cytb) gene a suitable candidate to study phylogenetic
evolution at the intra- and inter-species levels and target
for specific primers and probes (Brown et al. 1979; Xin
et al. 2006). These features grew our interest to design
In a preliminary phase of this investigation, speciesspecificity of the designed primers was assessed separately using DNA extracted from cat, dog, pig, monkey
and rat muscle tissues. PCR amplification was accomplished in a 25 µl total volume containing 0.5 U GoTaq
Flexi DNA Polymerase (Promega, Madison, USA), 5 µl
of 5X GoTaq Flexi Buffer, 200 µM each of dNTP,
1.5 mM MgCl2, 0.2 µM primers and 0.5 µl (20 ng/µl)
of total DNA in ABI 96-well verity thermal cycler
(Applied Biosystems, Foster City, CA) and the cycling
parameters were initial denaturation at 94°C for 3 min
followed by 35 cycles of denaturation at 94°C for 30 s,
annealing at 62, 62, 60, 62 and 61°C for cat, dog, pig,
monkey and rat primers, respectively, for 30 s and elongation at 72°C for 30 s and final elongation at 72°C for
5 min. The amplified products of species-specific simplex PCR were visualised first on 2% agarose gel
Table 2. Species-specific oligonucleotide primers for five target meat species.
Species
Genes
Primers
Primer sequence (5ʹ–3ʹ)
Amplicon size (bp)
Cat
Cytochrome b
ATPase 6
Pig
ND5
Monkey
ND5
Rat
ATPase 6
GGAATAATGTTTCGACCACTAAGC
TGCCTGAGATGGGTATTAGGAT
TGGCTCTAGCCGTTCGATTA
AAGGCAACAGCAAATTCTAGG
CCATCCCAATTATAATATCCAACTC
TGATTATTTCTTGGCCTGTGTGT
TGAGACCTCCAACAAATACTAGC
CTCTATGGCAGAAGGTAGTCAG
ATCATCAGAACGCCTTATTAGC
AGGTTCGTCCTTTTGGTGTA
172
Dog
Fwd
Rev
Fwd
Rev
Fwd
Rev
Fwd
Rev
Fwd
Rev
163
141
129
108
4
Md. A. Razzak et al.
1500
1000
[FU] Multiplex PCR of cat, dog, pork, monkey and rat
700
200
500
141 bp
150
400
163 bp
131 bp
100
300
172 bp
108 bp
50
200
150
0
15
Downloaded by [University of Malaya] at 19:41 19 October 2015
100
100
200 300
500 850
[bp]
50
15
L
1
2
3
4
5
6
7
8
9
10
11
Figure 1. (colour online) The gel image and the electropherograms of species-specific simplex and multiplex PCR. In the gel image: L,
Ladder; Lane 1–5, species-specific simplex PCR of cat (Felis catus), dog (Canis lupus familiaris), pig (Sus scrofa), monkey (Macaca
fascicularis) and rat (Rattus rattus) DNA from raw meat, respectively; Lane 6, multiplex PCR for five target meat species; Lane 7–11,
species-specific simplex PCR of heat-treated (121°C for 2.5 h) rat (Rattus rattus), monkey (Macaca fascicularis), pig (Sus scrofa), dog
(Canis lupus familiaris) and cat (Felis catus) meat DNA. The electropherogram of Lane 6 is demonstrated by respective labels.
stained with Florosafe DNA stain (1st Base
Laboratories, Selangor, Malaysia) using a gel image
documentation system (AlphaImager HP, California,
USA; data not shown) and second on gel-image and
electrochromatograms of Bio-Rad Experion Automated
Electrophoresis Station (Bio-Rad Laboratories, Inc.,
USA; Figure 1).
Multiplex PCR optimisation
The tetraplex PCR was carried out for cat, dog, monkey and
rat and finally multiplex PCR to detect cat, dog, pork, monkey and rat in ABI thermal cycler (Applied Biosystems,
Foster city, CA) in a 25 µl total volume containing 1 U
GoTaq Flexi DNA Polymerase (Promega, Madison, USA),
5 µl of 5X GoTaq Flexi Buffer, 200 µM each of dNTP,
1.5 mM MgCl2, 0.2–0.4 µM primers and 2.5 µl (0.5 µl of
20 ng/µl for each species) of total DNA with the cycling
parameters of initial denaturation at 94°C for 3 min following 35 cycles of denaturation at 94°C for 30 s, annealing at
59.5°C for 30 s and elongation at 72°C for 30 s and final
elongation at 72°C for 5 min. Negative template control of
PCR reaction (PCR reaction mixture without template DNA)
was included to ensure the purity of the PCR reaction mixture from contaminating DNA. The amplified PCR products
were analysed by Bio-Rad Experion Automated
Electrophoresis Station (Bio-Rad Laboratories, Inc.) using
Experion DNA 1 K Analysis Kit (Bio-Rad Laboratories,
Inc.). Typically, 1 µl of diluted (50 ng/µl) PCR products
was applied with 5 µl of specialised buffer into each of the
11 sample wells and 9 µl of gel-stain into gel-stain and
priming wells of the Experion DNA Chip. One microlitre
of DNA 1000 ladder containing 1500, 1000, 850, 700, 500,
400, 300, 200, 150, 100, 50, 25 and 15-bp marker DNAs was
applied into the ladder well. The samples were vortex-mixed
for 1 min at 2000 rpm and were immediately run on the
bioanalyser. The separation was achieved within 30 min by
the application of high voltage in the sieving polymer and
specialised buffer in the microfluidic channels through independent electrodes for each well and the automated electrophoretic patterns and the results were displayed as gel image
and electropherograms (Figures 1–4).
Results and discussion
Comparative perspectives of biomarker-based species
detection schemes
The authentication of meat species is of increasing
concern and vital to ensure quality food in compliance
with health, religion and fair prices (Rohman et al.
2011; Ali, Hashim, Mustafa, et al. 2012; Ali, Kashif,
Uddin, et al. 2012). Current analytical methods for
species identification are based on detecting lipid, protein or DNA biomarkers with identifying fingerprints
(Di Pinto et al. 2013; Von Bargen et al. 2013). The
principle of lipid-based methods includes the positional
analysis of fatty acid in triacylglycerol (TAG) and 2-
Food Additives & Contaminants: Part A
5
(a)
[FU]
1500
Multiplex PCR of target species in heat treated model
beef meatball spiked with 1% target meat species
200
1000
700
150
Pig
100
Dog
500
Monkey
50
Cat
Rat
400
0
15
100
200 300
500
850
[bp]
300
[FU]
Multiplex PCR of beef meatball spiked with 0.1%
target meat species
200
200
150
150
Pig
100
100
Monkey
Dog
Rat
Cat
50
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50
0
15
15
L
1
2
3
4
5
6
8
7
9
10
100
200
300
500
850
[bp]
11
(b)
[FU]
1500
200
Multiplex PCR of heat treated chicken meatball
spiked with 1% target meat species
1000
150
700
Pig
100
500
Monkey
Dog
Rat
Cat
50
400
0
15
300
100
200 300
500
850
[bp]
[FU]
200
Multiplex PCR of chicken meatball spiked with 0.1%
target meat species
200
150
150
Pig
100
100
Monkey
50
50
Rat
15
Dog
Cat
0
L
1
2
3
4
5
6
7
8
9
10
11
15
100
200 300
500
850
[bp]
Figure 2. (colour online) The gel image and the electropherograms of multiplex PCR (M-PCR) of beef (a) and chicken (b) meatballs
with sensitivity. In the gel images; L, Ladder; Lanes 1–5, species-specific PCR with multiplex optimisation to detect cat (Felis catus), dog
(Canis lupus familiaris), pig (Sus scrofa), monkey (Macaca fascicularis) and rat (Rattus rattus) DNA, respectively, spiked in beef and
chicken meatballs; Lane 6, multiplex PCR to detect five target species in beef and chicken meatball in raw state and Lane 7, in heattreated (121°C for 2.5 h) states; Lanes 8 and 9, M-PCR of beef and chicken meatball spiked with 0.5 and 0.1% target meat species,
respectively; Lane 10 of figure (a), M-PCR of heat-treated (121°C for 2.5 h) target meat species and Lane 10 of figure (b) and 11 of figure
(a), positive control (M-PCR of beef and chicken meatball in raw state); Lane 11 of figure (b), negative control. Electropherograms of
Lanes 7 and 9 show the detection of five meat species in heat-treated state and from 0.1% spiked target meat species, respectively.
monoacylglycerol (2-MAG; SzabÓ et al. 2007), as well
as the analysis of volatile organic compounds (VOCs;
Acevedo et al. 2011). However, these methods have
important limitations; for example, the content and varieties of fatty acids in TAGs and MAGs can be extensively modified during the cooking process (Marikkar
et al. 2011) and it is not sensitive enough to analyse the
food samples without extracting and pre-concentrating
the VOCs they contain (Ali, Kashif, Uddin, et al. 2012).
In the case of protein-based methods, quantification of
histidine dipeptides (Aristoy & Toldrá 2004), analysis of
sarcoplasmic muscle proteins such as parvalbumins
(Addis et al. 2010; Von Bargen et al. 2013), speciesspecific osteocalcin (Balizs et al. 2011), analysis of
single collagen peptide (Buckley et al. 2010), as well
as detection of species-specific proteins by enzymelinked immunosorbent assay (ELISA; Hsieh et al.
2002; Asensio et al. 2008) are in wide practices.
However, these methods not only need complicated
machineries and extensive analytical skills, but also
6
Md. A. Razzak et al.
(a)
[FU
1500
Multiplex PCR of heat treated beef burger
spiked with 1% target meat species
200
1000
150
700
Pig
100
Dog
Monkey
500
50
Rat
400
15
100 200
300
500
850
[bp]
[FU
300
Multiplex PCR of heat treated beef burger
spiked with 0.1% target meat species
200
150
200
Monkey Pig Dog
150
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Cat
0
Cat
100
Rat
100
50
50
0
15
15
L
1
2
3
4
5
6
7
8
9
10
100 200
300
500
850
[bp]
11
(b)
[FU
1500
Multiplex PCR of heat treated chicken burger
spiked with 1% target meat species
200
1000
150
700
100
Pig
500
50
400
Monkey
Dog
Rat
Cat
0
15
300
100 200
300
500
850
[bp]
[FU
200
Multiplex PCR of chicken burger spiked with
0.1% target meat species
200
150
150
100
100
50
Monkey Pig
Dog
Cat
50
Rat
15
0
L
1
2
3
4
5
6
7
8
9
10
11
15
100 200
300
500
850
[bp]
Figure 3. (colour online) The gel image and the electropherograms of multiplex PCR (M-PCR) of beef (a) and chicken (b) burger. In
the gel image: L, Ladder; Lanes 1–5, species-specific PCR with multiplex optimisation to detect cat (Felis catus), dog (Canis lupus
familiaris), pig (Sus scrofa), monkey (Macaca fascicularis) and rat (Rattus rattus) DNA, respectively, spiked with 1% target meat
individually in burger; Lane 6, multiplex PCR to detect five target species in burger in raw state and Lane 7, heat-treated (121°C for
2.5 h) state; Lanes 8 and 9, M-PCR of beef burger spiked with 0.5 and 0.1% target meat species, respectively; Lane 10, positive control;
Lane 11, negative control (M-PCR with no template). Electropherograms of Lanes 7 and 9 show the detection of five meat species in
heat-treated state and from 0.1% spiked target meat species, respectively.
provide conflicting outcomes (Ali, Kashif, Uddin, et al.
2012). Furthermore, sensitivity is greatly compromised
in proteomic approaches as given by Von Bargen et al.
(2013), who detected 0.55% horse or pork in a beef
matrix and 0.13% pork contamination in beef, which is
much lower than PCR-based techniques (0.00001 ng
Food Additives & Contaminants: Part A
(a)
7
[FU
Multiplex PCR of heat treated beef frankfurter
spiked with 1% target meat species
200
1500
1000
150
700
100
500
Pig
50
400
Monkey
Dog
Rat
Cat
0
15
100
200 300
500 850
[bp]
[FU
300
Multiplex PCR of beef frankfurter spiked with
0.1% target meat species
200
200
150
150
100
Pig
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Dog
Cat
Monkey
Rat
100
50
50
0
15
15
L
1
3
2
5
4
7
6
8
10
9
100
200 300
500 850
[bp]
11
(b)
[FU
1500
Multiplex PCR of heat treated chicken frankfurter
spiked with 1% target meat species
200
1000
150
700
100
Pig
500
50
400
Monkey
Dog
Rat
Cat
0
15
300
100
200 300
500 850
[bp]
[FU
200
200
Multiplex PCR of chicken frankfurter spiked
with 0.1% target meat species
Pig
150
Dog
150
Monkey
100
100
Cat
Rat
50
50
15
0
L
1
2
3
4
5
6
7
8
9
10
11
15
100
200 300
500 850
[bp]
Figure 4. (colour online) The gel image and the electropherograms of multiplex PCR (M-PCR) of beef (a) and chicken (b) frankfurter.
In the gel image: L, Ladder; Lanes 1–5, species-specific PCR with multiplex optimisation to detect cat (Felis catus), dog (Canis lupus
familiaris), pig (Sus scrofa), monkey (Macaca fascicularis) and rat (Rattus rattus) DNA, respectively, spiked with 1% target meat
individually in frankfurters; Lane 6, multiplex PCR to detect five target species in model beef frankfurter in raw state and Lane 7, heattreated (121°C for 2.5 h) state; Lanes 8 and 9, M-PCR of frankfurter spiked with 0.5 and 0.1% target meat species, respectively; Lane 10,
positive control; and Lane 11, negative control. Electropherograms of Lanes 7 and 9 show the detection of five meat species in heattreated state and from 0.1% spiked target meat species, respectively.
DNA in pure and 0.1% (w/w) of meat in complex
matrices; Rashid et al. 2015). Another drawback of the
“shotgun proteomics” is that after trypsin digestion of
the whole protein mixture, samples become much more
complex to analyse compared to individual protein
spots, thus making it necessary to use powerful mass
8
Md. A. Razzak et al.
Downloaded by [University of Malaya] at 19:41 19 October 2015
spectrometers together with high-throughput bioinformatics resources which are capable of analysing the
enormous amount of the generated MS/MS spectra
(Sentandreu & Sentandreu 2011). Moreover, cross-reactivity between closely related species may occur as has
happened in case of chicken and turkey (Sentandreu &
Sentandreu 2011). Furthermore, lipidomic and proteomic approaches require expensive instrumentations like
GC-MS, HPLC, e-nose, MALDI-TOF and Q-TOF mass
spectrometry for which skilled manpower is a must to
deal with the experiments and resulting analysis (Ali,
Kashif, Uddin, et al. 2012, Ali et al. 2014). On the other
hand, DNA and ubiquitous and robust biomarkers and
could be easily amplified from a single copy to millions
to ensure easy analysis using simple instrumentations
and comparatively fewer skill sets (Ali et al. 2011).
DNA extraction and PCR optimisation
To prevent DNA loss at aqueous and organic phase
separations (Karabasanavar et al. 2011), a spin columnbased extraction technique was used. This measure effectively increased the yield of good quality DNA. Because
DNA in heat-treated meat products is highly susceptible to
degradation, 100 mg commercial samples were used for
DNA extraction. The quantity and quality of total
extracted DNA was estimated based on absorbance at
260 nm and 280 nm in a UV-Vis Spectrophotometer
(Biochrom Ltd, Cambridge, UK). The A260/A280 ratios
of extracted DNA were between 1.7 and 2, which indicated a high quality of DNA in all specimens (Parchami
Nejad et al. 2014). Because higher annealing temperature
increases primer specificity and reduces non-specific PCR
amplification (Wu et al. 2009; Ali, Hashim, Mustafa, et al.
2012; Rahman et al. 2015), a repeated run of gradient
PCR (data not shown) was performed to optimise the
higher end of annealing temperatures for all simplex and
multiplex PCR assays.
After optimising the individual simplex PCR assays,
multiplex PCR systems were adjusted by sequential addition of cat, dog, pig, monkey and rat species as shown in
lane 6 of Figure 1. This eliminated the probability of
potential primer dimer or multimer formation
(Matsunaga et al. 1999; Zhang 2013). As the agarose gel
eventually fails (data not shown) to separate the products
of multiplex PCR systems with shorter-length differences
in amplified products, (Bottero & Dalmasso 2011), a
micro-fluidic-based capillary electrophoresis system incorporated in a bioanalyser was used to ensure accuracy,
precision, resolution (~10 bp) with minimum consumption
of samples and time (Chen et al. 2014; Rahman et al.
2015). Bio-Rad Experion Automated Electrophoresis
Station provided automated banding patterns along with
the electropherograms for all of the five targets amplified
in this multiplex system (Figures 1–4).
Species-specificity of designed primers
The specificity, precision and melting temperature (Tm)
of the primer play crucial role in the development of a
multiplex PCR because its success depends on the ability of the primers to be selectively annealed with their
respective targets under a single set of PCR conditions,
including reaction volume, cycling and annealing (Ali
et al. 2014). Because the presence of a single mismatch
in the primer binding site may reduce the efficiency of
the PCR assay or may lead to failure of PCR amplification (Wu et al. 2009), the estimation of oligonucleotide
mismatch is a key factor in designing species-specific
primers (Rahman et al. 2015). The primers designed
here contained 5–13 bp (23–45%) mismatches with
other relevant species and very closely spaced Tm
(61–62°C), ensuring the primers’ annealing only with
the DNA template of target species and not with any
non-target species (Matsunaga et al. 1999). Pairwise
distance analysis of primer sequences (data not shown)
against 15 animal and 5 plant species computed using
the maximum composite likelihood method (Tamura
et al. 2011) revealed a minimum distance between monkey and pigeon and maximum distance between rat and
tuna, reflecting very high genetic distance and very little
or no probability of cross-amplification of non-target
species (Ali et al. 2013).
In the PCR run, simplex PCRs were performed on
DNA extracted from muscle tissues of each target species to verify the specificity of the designed primers.
The species-specific primers amplified 172, 163, 141,
129 and 108 bp fragments from cat, dog, pig, monkey
and rat DNA template, respectively. The specificity of
the each pair of the designed primers was checked oneby-one and also against the 15 meat-providing and
expensive terrestrial (beef, chicken, goat, lamb, buffalo,
venison, duck, pigeon and quail) and aquatic species
(salmon, cod, tuna, carp, rohu and tilapia) and 5 plant
materials (wheat, tomato, garlic, onion and pepper)
which are commonly used in the preparation of meat
products. In each individual assay, each pair of primers
was first tested against the other four species of the
multiplex system as non-target DNA and then with the
most commonly used commercially important meat,
fish, and plant species and no cross-amplification was
detected (data not given).
All assays were carried out in triplicate on three
different days by three independent analysts to avoid
any operator bias or prejudice. The results indicated no
cross-amplification even on repetition in blind experiments. On the other hand, primer specificity of the
Food Additives & Contaminants: Part A
other species was also examined through multiplex PCR
optimised with DNA isolated from other non-target species (data not shown). Moreover, successful amplification
of DNA extracted from prolonged and severely heattreated (121°C for 2.5 h) pure meat samples through
both simplex (Figure 1; lanes 7–11) and multiplex
(Figure 2a; lane 10) PCR assays were attained.
Downloaded by [University of Malaya] at 19:41 19 October 2015
Specificity and sensitivity under complex food matrices
Adulteration often happens in commercial minced meat
products such as meatball, burger and frankfurter (Tanabe
et al. 2007; Ali et al. 2013; Rahman et al. 2014, 2015).
Therefore, the performance of the developed multiplex
PCR assay was tested under a complex background of
beef and chicken meatballs, burgers and frankfurters adulterated with 1%, 0.5% and 0.1% of target meats, both
individually and collectively. The prepared meat products
were autoclaved at 121°C at 45 psi for 2.5 h to reflect the
effects of heat treatment of processed foods.
In the case of individual target meat adulteration,
only specific target was amplified (Figure 2–4, lanes
1–5), suggesting the stringent specificity of the
designed primers. The DNAs of target meat species
from all dummy meat products containing as low as
0.1% adulteration were successfully amplified by use
of the developed multiplex PCR system (Figures 2–4;
lanes 6–9). The substitution of lower-valued meats
with higher-priced ones has long been done to gain
economic profits. However, the mixing of less than
1% low-priced meats does not make any significant
profit while weighing a great risk (Ali, Asing, Hamid,
et al. 2015). Thus, the detection of 1% adulteration was
considered sufficient to prove the sensitivity and reliability of multiplex PCR assays (Hou et al. 2014; Ali,
Razzak, Hamid, et al. 2015). Consequently, we spiked
1% minced meat with dummy meat products
(Figure 2–4, lanes 1–5). However, some researchers
have carried out multiplex PCR to detect 0.1% adulteration (Safdar et al. 2014; Safdar & Junejo 2015).
Therefore, we also tested the sensitivity of the developed assay with 0.5% and 0.1% adulteration in various
meat products (Figure 2–4, lanes 8–9).
An extensive literature search demonstrated the previously developed multiplex PCR assays targeted relatively longer amplicons (≥200 bp) and in most of the
cases, the assays were not tested under food-processing
conditions and complex matrices of commercial meat
products. Recent studies showed that ≤150 bp amplicons
have a higher chance of survival in degraded samples and
thus offer better sensitivity and method validity
(Rodriguez et al. 2004; Cammà et al. 2012; Rahman
et al. 2015). Lin and Hwang (2008) reported that highly
degraded DNA could not be detected if ≥300 bp amplicons are used.
9
Furthermore, earlier multiplex PCR assays included
only pig and horse meats among the haram (not allowed)
meat species (Matsunaga et al. 1999; Di Pinto et al. 2005;
Kitpipit et al. 2014; Safdar et al. 2014), which have
limited scope in halal authentication. Additionally, the
validity of previously developed multiplex PCR assays
was determined by analysing one dummy meat product
(Hou et al. 2014; Parchami Nejad et al. 2014; Safdar et al.
2014; Safdar & Junejo 2015). We tested here five different
beef and chicken meatballs, eight burgers and seven frankfurters spiked with target meats to validate the assay.
Analysis of commercial meat products
The robustness, validity and reliability of the developed
multiplex assay was determined by screening commercially available popular meat products such as beef and
chicken meatballs, burgers and frankfurters, which are
widely consumed across Malaysia, Indonesia, China and
most parts of the world (Rohman et al. 2011; Ali, Hashim,
Mustafa, et al. 2012; Ali et al. 2013; Ali, Razzak, Hamid,
et al. 2015; Rahman et al. 2014; Rahmania et al. 2015). To
fulfil the selected goal, five different halal branded beef
and chicken meatballs, eight burgers and seven frankfurters of different manufacturers were collected and tested
from three different Malaysian outlets on three different
days. The statistical data of the tested samples are given in
Table 3. While in all model meatballs with deliberate
adulteration the target species were positively detected in
blind experiments, all commercial samples were found
with negative targets. All of these experiments were carried out in triplicate, by three different analysts on three
different days to avoid any prejudice.
The finding of this paper of non-fraudulent labelling of
pig, dog, rat, cat and monkey in commercial products was
against the conventional wisdom because fraudulent labelling is quite common around the globe (Fajardo et al. 2010;
Doosti et al. 2014). However, in Malaysian perspectives it
was not a surprise because the Malaysian government is
highly committed to develop “Halal Hub” and has been
strictly monitoring local markets from time to time. Thus,
the study finding was logical and suggested the halal sanctity of Malaysian meatballs, burgers and frankfurters.
Conclusion
Adulterated meat in the food supply chain impacts on
public sentiments, and jeopardises health and religious
faiths. Further, rat, cat, dog and monkey are potential
carriers of plague, ringworm, hydrophobia (rabies), and
herpes virus (Herpevirus simiae), simian virus 40
(SV40) and other zoonotic diseases and hence are not
safe and hygienic for public consumption. Religion has
always been a key determinant in meat consumption,
such as pork consumption which is prohibited in Islam
10
Md. A. Razzak et al.
Table 3. Wide screening of model and commercial ready to eat meat products sold in markets using developed multiplex PCR.
Detected species
Downloaded by [University of Malaya] at 19:41 19 October 2015
Meat products
Different meat products spiked with
Pig meat
Dog meat
Cat meat
Monkey meat
Rat meat
Beef meatballs spiked with all target meat species (raw)
Beef meatballs spiked with all target meat species (heat-treated)
Chicken meatballs spiked with all target meat species (raw)
Chicken meatballs spiked with all target meat species (heat-treated)
Beef burgers spiked with all target meat species (raw)
Beef burgers spiked with all target meat species (heat-treated)
Chicken burgers spiked with all target meat species (raw)
Chicken burgers spiked with all target meat species (heat-treated)
Beef frankfurter spiked with all target meat species (raw)
Beef frankfurter spiked with all target meat species (heat-treated)
Chicken frankfurter spiked with all target meat species (raw)
Chicken frankfurter spiked with all target meat species (heat-treated)
Commercial meat products*
Beef meatball
Chicken meatball
Beef burger
Chicken burger
Beef frankfurter
Chicken frankfurter
Pig
Dog
Cat
Monkey
Rat
PCR accuracy (%)
54/54
–
–
–
–
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
–
54/54
–
–
–
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
–
–
54/54
–
–
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
–
–
–
54/54
–
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
–
–
–
–
54/54
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
9/9
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
0/9
100
100
100
100
100
100
Notes: *Five different brands of each beef and chicken meatballs, 8 burgers and 7 frankfurter items were analysed to validate the developed multiplex PCR
assay. The numerator and denominator of each fraction denote the number of positive detection and total number of samples analysed using the multiplex
PCR assay.
and Judaism. Some incidents such as food allergy due
to pork consumption and porcine DNA in meat pies and
pasties supplied to the prisons of the UK have greatly
promoted the authentication of porcine derivatives in
various meals and menus. Additionally, Islamic law
has prohibited Muslims from eating animals having
canine teeth or fangs such as dog, cat, monkey and
rat. Furthermore, the availability of stray dogs and cats
in certain countries has made meat from these species a
potential adulterant in costly meats such as beef, lamb
and chicken. On the other hand, monkey (macaque) is a
widespread species and often can be hunted free of
charge. Thus, the chances of its falsification are very
high. Carefully designed species-specific PCR under
optimised conditions is conclusive to detect and identify
species, eliminating the need for restriction digestion
and/or sequencing of PCR products. Instead of single
species, the detection of five different species in a single
assay platform clearly cut the analysis cost by at least
five times, offering easy analysis of food components in
the industry and retail outlets. The multiplex PCR systems developed here conveniently detected five meat
species forbidden in Islamic foods from spiked meat
products with as low as 0.1% adulteration, clearly
showing its appeal in the halal food industry and halal
regulatory bodies. Shorter amplicon lengths (108–172
bp) and successful amplification of target species
under raw and extremely heat-treated meat species suggested that the method would be used in the screening
of target species under compromised states and complex
matrices. In addition to pentaplexing, the assay could be
used in simplex, duplex, triplex, and tetraplex PCR
systems based on the requirements and needs.
Acknowledgements
The authors acknowledge wildlife Malaysia and Dewan
Bandaraya Kuala Lumpur (DBKL) for providing monkey, dog
and cat meat samples.
Ethical compliance
The ethical permission was obtained from the University of
Malaya Ethical Committee (ref. no.: NANOCAT/23/07/2013/
MEA(R)) and all the animals and meats used in this study
were handled following the institutional and national guideline
for animal care.
Disclosure statement
No potential conflict of interest was reported by the authors.
Food Additives & Contaminants: Part A
Funding
This work was supported by University of Malaya Research
[grant number GC001A-14SBS] to M.E. Ali.
Downloaded by [University of Malaya] at 19:41 19 October 2015
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