Beruflich Dokumente
Kultur Dokumente
Meat Science
journal homepage: www.elsevier.com/locate/meatsci
Nanotechnology and Catalysis Research Centre, Universiti Malaya, Kuala Lumpur 50603, Malaysia
Institute of Halal Products Research, Universiti Putra Malaysia, Serdang 43400, Malaysia
Institute of Nanoelectronic Engineering, Universiti Malaysia Perlis, Kangar 01000, Perlis, Malaysia
a r t i c l e
i n f o
Article history:
Received 14 June 2013
Accepted 19 March 2014
Available online 26 March 2014
Keywords:
Canine derivatives
Meatballs
Extensive autoclaving
Halal food industry
a b s t r a c t
A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was
developed. The assay selectively amplied a 100-bp region of canine mitochondrial cytochrome b gene from
pure, raw, processed and mixed backgrounds. The specicity of the assay was tested against 11 animals and 3
plants species, commonly available for meatball formulation. The stability of the assay was proven under
extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef
meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using
0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of
the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in
processed foods.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
The prospects for Halal meats and meats products are rapidly
expanding (Ali, Hashim, Dhahi, Mustafa, Man, et al., 2012). Currently,
halal food consuming population has reached to 1.8 billion and the
turnover of halal market has exceeded USD 661 billion (Ali, Kashif,
et al., 2012). Several factors including increasing workloads are pushing
potential halal consumers to spend more time in workplaces, leaving no
time for self-cooking. Thus a growing number of people are increasingly
being forced to eat readymade foods such as burger, pizza, hot dogs,
meatball, soups and so on (Ali, Kashif, et al., 2012). Due to the specialized preparation of halal meats, the prices for halal brands are higher,
especially in those countries where Muslims are the minority. Thus
the fraudulent labelling of halal brands is prevalent (Ali, Hashim,
Mustafa & Che Man, 2012). To cope up with the situation demands
and business requirements, many countries including Malaysia,
Indonesia, Thailand, Singapore, China, Brazil, Australia and Newzealand
are having regulatory bodies to protect the sanctity of Halal food
markets (Ali, Kashif, et al., 2012). Thus the food manufacturers, marketers and regulators need innovative, easily performable and
improved authentication techniques for the verication of halal brands.
Meatballs made up with comminute meats are very popular
throughout the world including Malaysia, Indonesia, China, Vietnam,
Corresponding author. Tel.: +60 3 7967 6955; fax: +60 3 7967 6956.
E-mail address: eaqubali@gmail.com (M.E. Ali).
http://dx.doi.org/10.1016/j.meatsci.2014.03.011
0309-1740/ 2014 Elsevier Ltd. All rights reserved.
India, the USA and the Europe (Ali, Hashim, Mustafa, Che Man, Dhahi,
et al., 2012; Rohman, Sismindari, Erwanto, & Che Man, 2011). Dog
meat is a potential adulterant in halal foods since stray dogs are available in many countries without any offered prices. Reports have been
made for the consumption of dog meats in certain countries such as
Vietnam, South Korea and China (Bartlett & Clifton, 2003; Podberscek,
2009). Foreign workers, especially from Myanmar and Vietnam origins,
are reported to consume stray dog meat in Malaysia. However, no survey is made to verify the mixing of dog meats in commercial meat products across Malaysia or elsewhere in the world. The mixing of dog origin
materials in food is a serious issue in many religions including Islam and
Buddhism (Khattak et al., 2011; Mahanarongchai & Marranca, 2007).
Several methods such as SYBR green real-time PCR (Farrokhi & Jafari
Joozani, 2011), molecular beacon real-time PCR (Yusop, Mustafa, Che
Man, Omar, & Mokhtar, 2012), TaqMan probe real-time PCR (Ali,
Hashim, Dhahi, et al., 2012), electronic nose coupled with gas chromatographymass spectrometry (Nurjuliana, Che Man, Mat Hashim, &
Mohamed, 2011), Fourier transform infrared spectroscopy (Rohman
et al., 2011), enzyme-linked immunosorbant assay (Asensio, Gonzlez,
Garca, & Martin, 2008), PCR-RFLP (Ali, Hashim, Mustafa, & Che Man,
2012) and nanoparticle sensors coupled with optical or uorescence
spectroscopy (Ali et al., 2011) have been proposed for the authentication of meat species. Although cumbersome to some extent, the conventional species specic PCR assay is an easily affordable and reliable
method for the routine analysis of animal meat products in food industry (Arslan, Ilhak, & Calicioglu, 2006; Matsunaga et al., 1999). For dog
405
dog meat contaminated meatballs 1%, 0.5%, 0.2%, 0.1%, and 0.01% of dog
meat were added with 100 g of chicken and beef meat in the formulation. The meats with all other ingredients were mixed well by vigorous
blending and the emulsied homogenous meat mixtures were mechanically given into ball shape. To simulate cooking and extensive autoclaving effect thus prepared raw meatballs were subjected to cooking at 100
C for 90 min and autoclaved at 120 C under 45-psi pressure for 2.5 h.
All samples were prepared on three different days by three independent
analysts and were stored at 20 C for DNA extraction.
Chicken meatball
a
100 g
7.5 g
5g
1.25 g
0.14 g
0.01 l
3.28 g
0.05 g
Beef meatball
a
100 g
7.5 g
5g
1.5 g
1.25 g
1.25 g
2.5 g
0.05 g
Dog meatball
100 g
7.5 g
5g
1.5 g
1.25 g
1.25 g
0.14 g
0.01 l
3.28 g
2.5 g
0.05 g
a
1%, 0.5%, 0.2%, 0.1% and 0.01% of dog meat were mixed with a balanced amount of
chicken and beef meat to make 100 g specimen of each meatball meat.
406
Beef
Dog
The canine specicity of the assay was tested in three steps. In step 1,
the retrieved DNA sequences were aligned along with the primer
sequences using mega 5 (Tamura et al., 2011) software and clustalW
alignment tool for mismatch detection. A 3D plot was built using the
number of mismatches and pairwise distance data to nd the discriminating properties and molecular orbit of the primers using XLSTAT
version 2013.1.01 (Addinsoft, 2013). In step 2, cross species testing
was performed in a real PCR run using DNA extracted from the potential
meat and plant species for meatball preparation. Finally, in step three,
PCR product was puried using QIAquick PCR purication kit (Qiagen,
USA) and sequencing result was analyzed using the BLAST local
alignment tool in NCBI data base to conrm the canine cytb gene
specicty. The sequencing was done by the 1st Base Laboratories Pte.
Ltd., Selangor, Malaysia.
Sheep
Goat
Buffalo
Pig
Cat
Onion
Chicken
Quail
Tomato
Turkey
Duck
Garlic
Fig. 1. Phylogenetic tree build by the neighbor-joining method using 100 bp primer
binding region of cytb/cob-gene sequences of dog and other 13 animals and plant species
showing evolutionary distance between dog and other species potential for meatball
formulation.
data base. Complete identity was found only with the canine cytb
gene. In real PCR run, the assay amplied only 100 bp canine cytb
gene target (Fig. 3). Sequencing of PCR product also conrmed the
specicity for the canine cytb gene.
It has been quoted in several reports that the efciency of the PCR
assay might reduce or end up with PCR amplication failure because
of the presence of single mismatches in the primer binding region
(Ali, Hashim, Mustafa, & Che Man, 2012; Smith, Vigilant, & Morin,
Fig. 2. 3D plot showing the discrimination of dog specic target using the number of
mismatches in the primer binding sites and pair wise distances. Here the X axis represents
pair wise distance, and the Y and Z axes represent the number of forward and reverse
primer mismatches between dog and other potential animal and plant species for
meatball preparation.
407
Fig. 3. Species specicity analysis showing a 100 bp PCR product of canine meat DNA (Lane 1). Lane 1: dog DNA, Lanes 214: DNA from chicken, turkey, duck, quail, sheep, goat, beef,
buffalo, pig, cat, tomato, garlic and onion respectively; Lane N: negative control.
2002; Wu et al., 2009). The proposed mitochondrial cytochrome b (mtcytb) based primer pairs had perfect matching with dog and multiple
mismatches (N5 nucleotide) in the primer binding sites with other 13
potential species for meatball preparation and in real PCR run 100%
canine specicity was conrmed. Previously, different PCR assays
using mitochondrial whole genome (322 bp) (lhak & Arslan, 2007),
cytb (808 bp) (Abdel-Rahman et al., 2009; Abdulmawjood et al.,
2003), D-loop (213 bp) (Gao et al., 2004) and 12S rRNA (101 bp)
(Martn et al., 2007) have been proposed for dog meat detection. However, four of these assays used longer sized amplicon ( 213 bp)
(Abdel-Rahman et al., 2009; Abdulmawjood et al., 2003; Gao et al.,
2004; lhak & Arslan, 2007), which may breakdown under extensive
autoclaving and harsh processing conditions (Ali et al., 2011; Rojas
et al., 2010 Recently, short amplicon-length PCR assays are appreciated
for the authentication of meat-species in processed foods (b 150 bp)
(Ali, Hashim, Mustafa, & Che Man, 2012; Rojas et al., 2010). Martn
et al. (2007) proposed a PCR assay targeting 101 bp region of the canine
12S rRNA gene which should be suitable for the analysis of processed
foods. However, the latest cytb-based species identication scheme
showed more accurate reconstruction of mammalian phylogeny with
higher resolution (Nicolas et al., 2012). This is probably because of the
higher rate of intra-species conservation and protection of the cytb
gene over its counterparts. Hence, we speculated that mt-cytb based
short amplicon length (100 bp) PCR assay would give better detection
of canine derived materials from raw as well highly processed
meatballs.
composition, which is an undened method for concentration expression, can be manipulated through the use of higher amount of template
DNA. Furthermore as illustrated earlier for extensive process food analysis, certain literatures reported for the better sensitivity and stability of
the short amplicon based PCR assays over the longer ones (Ali, Hashim,
Mustafa, & Che Man, 2012; Rojas et al., 2010). Hence, the sensitivity of
the PCR-RFLP assay documented by Abdulmawjood et al. (2003) cannot
be higher than the present one and Martn et al. (2007).
Fig. 4. Specicity and sensitivity of canine specic PCR assay under chickendogwheat
our ternary mixture. A 100 bp PCR product was found from 1 to 0.1% of dog meat
contaminated ternary mixtures (Lanes 14) after extensive autoclaving. Lane L: 100 bp
ladder; Lanes 15: 1%, 0.5%, 0.2%, 0.1% and 0.01% dog meat mixed with chicken and
wheat our respectively; Lane N: negative control.
408
Fig. 5. Specicity and sensitivity of canine specic PCR assay under meatball matrices. A 100 bp PCR product was obtained from 1 to 0.2% of dog meat contaminated chicken meatball under
autoclaved, boiled and raw meatballs. Lane L: 100 bp ladder; Lanes 1 to 5: autoclaved; Lanes 6 to 10: boiled and Lanes 11 to 15: raw meatball samples contaminated with 1%, 0.5%, 0.2%, 0.1%
and 0.01% dog meat, respectively; Lane N: negative control.
were used as a positive control for screening the halal logo containing
commercial chicken and beef meatballs obtained from various super
markets across Malaysia. In a blind experiment, ve commercial brands
(AE) of chicken and beef meatballs were screen in triplicates on three
different days against the 0.2% positive control. While all positive
controls amplied the selective PCR products specic for canine cytb
gene, no commercial samples were found to be positive (Table 2 and
Fig. 6).
Although ve different PCR assays were previously proposed for
canine meat detection (Abdel-Rahman et al., 2009; Abdulmawjood
et al., 2003; Gao et al., 2004; lhak & Arslan, 2007; Martn et al., 2007)
none of them were tested for any commercial meat products. Therefore,
we analysed our assay performance in pure and contaminated state of
commercial meatballs. The presence of various additives and inhibitors
in commercial meat and food products might prevent the primer binding at specic sites and reduce the amplication efciency, diminishing
the sensitivity and specicity of a PCR assay (Bottero, Civera, Anastasio,
Turi, & Rosati, 2002; Calvo, Zaragoza, & Osta, 2001; Di Pinto, Forte,
Conversano, & Tantillo, 2005). Hence, a constant detection limit of
0.2% (0.04 ng DNA) was obtained in all meatball positive control,
demonstrating the performance of our PCR assay under the complex
background of meatball matrices. The Malaysian government has a
strong commitment to build a halal hub in local and international
domain and the Malaysian department of standards is responsible for
halal authentication across Malaysia (Talib, Ali, Anuar, & Jamaludin,
2008). Therefore, our study was in line with the government policy
and we found the validity and applicability of our assay for the detection
of canine tissues both in raw and processed commercial halal products.
4. Conclusion
A short amplicon length (100 bp) cytochrome b based conventional
PCR was documented for the detection of dog meat in commercial
meatballs. The specicity test against 14 different species demonstrated
that the assay was specic only for the dog cytb gene. Assessment under
raw, ternary admixtures, meatball matrices along with cooking and extensive autoclaving condition, reected that the method was suitable
for the analysis of raw as well as processed meats. A blind experiment
performed in triplicates on three different days by independent analysts, detected canine DNA only in positive controls. Screening of commercial meatballs from Malaysian supermarkets did not nd any
traces of canine tissues. The detection limit of the assay was 0.2% of
dog meat (0.04 ng Dog DNA) from a complex DNA pool of meatball.
5. Ethical statement
Ethical clearance was obtained from Institutional Animal Care and
Use Committee, University of Malaya (UM IACUC), Malaysia; (Reference
number: NANOCAT/25/04/3013/MMR (R) and the meat samples were
handled following the guideline for the proposed research.
Table 2
Meatball analysis using canine mt-cytb (100 bp) based PCR assay.
Meatball sample
Day 1
Day 2
Day 3
3
3
3
9
9
3
3
3
9
9
3
3
3
9
9
0/9
0/9
9/9
27/27
27/27
100
100
100
100
100
100
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0/9
0/9
0/9
0/9
0/9
100
100
100
100
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0/9
0/9
0/9
0/9
0/9
100
100
100
100
100
409
Fig. 6. Screening of commercial meatballs using canine specic PCR assay. No PCR product was obtain from commercial samples. Lane L: 100 bp DNA Ladder; Lanes 12: 0.2% dog meat
contaminated chicken and beef meatballs (positive control); Lanes 37: commercial chicken meatballs (AE); Lanes 812: commercial beef meatballs (AE) and Lane N: negative control.
Acknowledgment
This research was supported by the Universiti Malaya Research
Grant (RG153-12AET) to M.E. Ali and Malaysian Ministry of Higher Education MIS scholarship (KPT.B.600-18/3 Vol 2 133) to M.M. Rahman.
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