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Meat Science 69 (2005) 47–52

MEATSCIENCE

Analysis of raw meats and fats of pigs using polymerasechain reaction for Halal authentication

A.A. Aida a, Y.B. Che Man a,*, C.M.V.L. Wong b, A.R. Raha b, R. Son b

a Department of Food Technology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiab Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Received 18 February 2004; received in revised form 8 June 2004; accepted 8 June 2004

Abstract

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved

region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted

using Qiagen DNeasy� Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard

was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360

base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-

specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent re-

sults for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for

Halal authentication.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Pork; Lard; Cytochrome b; PCR; RFLP

1. Introduction

Consumers are concerned by a variety of issues, such

as food authenticity and adulteration. This has resulted

in increased awareness regarding the composition of

food products. The identity of the ingredients in proc-

essed or composite mixtures is not always readily appar-

ent and verification that the components are authentic

and from sources acceptable to the consumers may be re-

quired (Lockley & Bardsley, 2000). In most countries,food manufacturers choose to use lard as a substitute in-

gredient for oil because it is cheaper and easily available.

Pork and lard are serious matters in the view of some re-

ligions such as Islam and Judaism. Biological complica-

tions and health risks may be associated with daily

0309-1740/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.meatsci.2004.06.020

* Corresponding author. Tel.: +60-3-89468413; fax: +60-3-

89423552.

E-mail address: yaakub@fsb.upm.edu.my (Y.B. Che Man).

intake. In Islam, foods containing pig sources are Haram

(unlawful or prohibited) for Muslims to consume.Hence, it is an important task for food control labo-

ratories to be able to carry out species differentiation of

raw materials to be used for industrial food preparation

and the detection of animal species in food products.

This is especially crucial for Halal (lawful or permitted)

authentication, of food products. In order to protect

consumers from fraud and adulteration several analyti-

cal approaches have been made to identify animal spe-cies in food products. Methods have been developed

based on electrophoresis (Babiker, Glover, & Lawrie,

1981; Kim & Shelef, 1986), isoelectric focusing (Jaussen,

Hagele, Voorpostel, & de Baaij, 1990; King, 1984), chro-

matography (Ashoor, Moute, & Stiles, 1988; Saeed, Ali,

Abdul Rahman, & Sawaya, 1989), deoxyribonucleic

acid (DNA) hybridization (Chikuni, Ozutsumi,

Koishikawa, & Kato, 1990; Ebbehøj, 1991), polymerasechain reaction (PCR) (Meyer, Hofelein, Luthy, &

48 A.A. Aida et al. / Meat Science 69 (2005) 47–52

Candrain, 1995) and enzyme-linked immunosorbent as-

say (ELISA) (Chen, Hsieh, & Brigdman, 1998; Hsieh,

Johnson, Wetzstein, & Green, 1996).

In addition to protein-based techniques, DNA tech-

niques have become very important and are widely used

nowadays. It cannot be denied that there are several ad-vantages of DNA analysis methods: DNA is a relatively

stable molecule allowing analysis of processed and heat-

treated food products (Beneke & Hagen, 1998; Unseld,

Beyermann, Brandt, & Hiesel, 1995). DNA carries an

organism�s genetic information, and the information

content of DNA is greater than protein due to the de-

generacy of the genetic code as one goes from DNA to

protein (Wolf, Burgener, Hubner, & Luthy, 2000).DNA is a remarkably stable molecule allowing its ex-

traction from all kinds of tissue due to the ubiquity of

DNA in every type of cell (Wolf & Luthy, 2001; Wolf

et al., 2000).

PCR proved to be an adequate technique for detec-

tion of small amounts of DNA, specifically amplifying

a target region of template DNA in a rapid and sensi-

tive manner (Saiki et al., 1988). Many sequences of mit-ochondrial (mt) (e.g. cytochrome b (cyt b) gene)

(Burgener & Hubner, 1998; Matsunaga et al., 1999) or

genomic DNA have been analysed for various animals

like fish (Cespedes et al., 1999; Quinteiro et al., 1998),

game species (Wolf, Rentsch, & Hubner, 1999b) and

several domestic animals and from caviar (Birstein,

Doukakis, Sorkin, & DeSalle, 1998; Wolf, Hubner, &

Luthy, 1999a) by using either specific or universal prim-ers for inter- and intraspecies relationships in order to

establish the molecular phylogeny (Birstein & Desakke,

1998).

Compared to other techniques for species identifica-

tion by DNA-based methods, polymerase chain

reaction-restriction fragment length polymorphism

(PCR-RFLP) analysis of mt-DNA has offered the great-

est advantage (Bellagamba, Moretti, Comincini, & Val-fre, 2001). The advantages of PCR-RFLP are many: one

universal PCR-primer system in combination with a few

restriction enzymes (RE) can be sufficient for species

identification (Meyer et al., 1995). No references are nec-

essary once the restriction patterns of the species of in-

terest have been determined. A careful selection of RE

prevents ambiguous results caused by intraspecies poly-

morphisms (Wolf et al., 1999b). PCR-RFLP constitutesa simpler alternative to sequencing for the identification

of genetic variation between and within species (Borgo,

Souly-Crosset, Bouchon, & Gomot, 1996). The analysis

of PCR-RFLP of cyt b fragments has already been suc-

cessfully applied for species differentiation in heated and

processed meat products e.g. sausages (Meyer et al.,

1995).

In this study, mt-DNA was chosen as the target of in-vestigations. The objective of this study is to develop a

method for species identification from pork and lard

samples using PCR-RFLP analysis of a conserved re-

gion in the mt cyt b gene.

2. Materials and methods

2.1. Samples

Meat and fat samples from sheep, cow, chicken and

pig were used. Sheep, cow and chicken samples wereutilized as controls in this study. The samples were

purchased from a wet market in Sri Kembangan, Peta-

ling Street and Pasar Borong Selangor, Malaysia. The

samples were stored at �20 �C before the extraction

of DNA to prevent the enzymatic degradation of

DNA.

2.2. DNA extraction

DNA was extracted from 25 mg of meat and fat sam-

ples using the DNeasy� Protocol for animal tissue pro-

vided with the DNeasy� Tissue Kit (Qiagen, Hilden,

Germany). Approximately 25 mg of meat and fat sam-

ples was blended using a blender (Braun AG, Frankfurt,

Germany), placed in a 1.5 ml microcentrifuge tube. One

hundred and eighty microlitres ATL buffer and 20 llProteinase K were added and mixed by vortexing. The

mixture was incubated at 55 �C in a water bath to dis-

perse the sample overnight until the tissue was completely

lysed. The following day, 4 ll of RNase A (100 mg/ml)

was added and incubated for 2 min at room tempera-

ture. The mixture was mixed by vortexing for 15 s.

Two hundred microlitres AL buffer was added to the

sample, mixed thoroughly by vortexing and incubatedat 70 �C for 10 min. Two hundred microlitres ethanol

(96–100%) was added to mixture and mixed by vortex-

ing to yield a homogenous solution. The homogenous

solution was pipetted into the DNeasy� mini column sit-

ting in a 2 ml collection tube. The homogenous solution

was spun at 12,000g for 1 min. The flow-through and

collection tube was discarded and the DNeasy� mini

column was placed in a new 2 ml collection tube. Fivehundred microlitres AW1 buffer was added and spun

at 12,000g for 1 min. The flow-through and collection

tube was discarded and the DNeasy� mini column was

placed in another 2 ml collection tube. Five hundred

microlitres AW2 buffer was added and spun at full speed

for 3 min to dry the DNeasy� membrane and then the

flow-through and collection tube was discarded. The

DNeasy� mini column was placed in a clean 1.5 mlmicrocentrifuge tube. One hundred and fifty microlitres

AE buffer was pipetted directly onto the DNeasy� mem-

brane and incubated at room temperature at 1 min. This

was then spun at 12,000g for 1 min to elute. The DNA

solution was stored at 4 �C.

A.A. Aida et al. / Meat Science 69 (2005) 47–52 49

2.3. Oligonucleotide primers

A pair of primers was employed in PCR reaction. The

PCR primers used were CYTb1 (5 0-CCA TCC AAC

ATC TCA GCA TGA TGA AA-3 0) and CYTb2 (5 0-

GCC CCT CAG AAT GAT ATT TGT CCT CA-3 0),which were published by Kocher et al. (1989).

2.4. Polymerase chain reaction amplification of the

cytochrome b gene

Amplification of the mt cyt b gene was performed in a

final volume of 25 ll containing 30 ng of extracted DNA

which was measured by a spectrophotometer (A 260nm) (Beckman Coulter, CA, USA), 1x PCR reaction

buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.3), 25

mM MgCl2, 10 mM dNTPs, 10 pmol of each primer

and 5 units/ll of Taq DNA polymerase (Finnzymes,

Espoo, Finland). Amplification was performed with a

Perkin–Elmer (Gene Amp PCR system 2400) thermal

cycler according to the following PCR step-cycle pro-

gram: pre-denaturation of 94 �C for 2 min to completelydenature the DNA template, followed by 35 cycles of

denaturation at 94 �C for 5 s, annealing at 55 �C for

30 s, and extension at 72 �C for 40 s. Final extension

at 72 �C for 2 min followed the final cycle for complete

synthesis of elongated DNA molecules. Ten microlitres

of PCR products were electrophoresed at constant volt-

age (74 V) on 2% agarose gel (Promega, Madison, USA)

for about an hour in 1x TAE buffer, pH 8.0 and stainedby ethidium bromide. A 100 bp DNA ladder (Promega,

Madison, USA) was used as size reference. The gel

photo was taken using the Syngene gel documentation

system.

Fig. 1. Electrophoresis analysis of DNA extraction from meat and fat sampl

and 7, pork; 8, mutton fat; 9, beef fat; 10, chicken fat; 11, 12, 13 and 14, la

2.5. Restriction fragment length polymorphism analysis

Five units/ll of RE BsaJI (New England Biolabs,

Beverly, USA) were applied to 15 ll of amplified

DNA in a final volume of 20 ll digestion mixture [con-

taining 1x reaction buffer (10 mM Tris–HCl, 50–100mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol)]

and were incubated at 60 �C overnight for optimal re-

sult. Ten microlitres of the digested samples were elec-

trophoresed at constant voltage (100 V) on 2% agarose

gel (Promega, Madison, USA) for about an hour in 1x

TAE buffer, pH 8.0 and stained by ethidium bromide.

A 100 bp (Promega, Madison, USA) and 1 kb plus

DNA ladder (New England Biolabs, Beverly, USA)was used as size reference. The gel photo was taken us-

ing the Syngene gel documentation system.

3. Results and discussion

We employed a three step analysis to determine the

identity of meat and fat samples: (i) genomic and mt-DNA isolation (ii) mt-DNA is subjected to PCR ampli-

fication of the cyt b gene and (iii) the cyt b amplicon is

cut with RE to reveal the RE cutting pattern so that

the identity of the meat and fat source can be revealed.

Additionally, we wanted to determine if enough

DNA could be obtained from fats to carry out PCR am-

plification.

The quality of the extracted DNA from 25 mg ofmeat and fat samples using the DNeasy� Protocol for

animal tissue provided with the DNeasy� Tissue Kit

(Qiagen) was examined by electrophoretic analysis

through a 1.2% agarose gel (Promega). A band of high

intensity appeared in the lanes (Fig. 1). This showed that

es. M-1 kb plus DNA ladder; 1, mutton; 2, beef; 3, chicken meat; 4,5,6

rd.

50 A.A. Aida et al. / Meat Science 69 (2005) 47–52

a significantly high yield genomic and mt-DNA extracted

from meat and fat samples can be used as template for

PCR amplification of the cyt b.

Agarose gel electrophoresis of the PCR amplified

products (Fig. 2) from the samples resolved a band of

approximately 360 bp for the detection of cyt b gene.The result shows that the meat and fat samples pro-

duced enough mt-DNA for PCR amplification of the

cyt b gene. The amplification of an approximately

360 bp fragment is consistent with the results reported

by Meyer et al. (1995).

The CYTb1/CYTb2 primers employed are consid-

ered to be universal (Kocher et al., 1989) and were

shown to amplify DNA from the sample, which com-prises a considerable span of vertebrate evolutionary

Fig. 3. BsaJI restriction profile of cytochrome b PCR products amplified from

chicken meat; 4,5,6 and 7, pork; 10, chicken fat; 11,12,13 and 14, lard; M2-

Fig. 2. Electrophoresis analysis of cytochrome b PCR products amplified fro

chicken meat; 4,5,6 and 7, pork; 8, mutton fat; 9, cow fat; 10, chicken fat; 1

diversity. An advantage in employing universal primers

is that it obviates the requirement for an internal con-

trol, which is otherwise used to monitor the success of

DNA amplification (Partis et al., 2000).

PCR is a highly sensitive method, which had been

proven by the successful amplification of amplicons.The primers used were very specific amplifying only a

single band of expected size regardless of the concentra-

tion of template mt-DNA extracted from meats and

fats. Negative controls (PCR reaction mixtures without

template DNA) were always employed to make sure

there was no contamination in the PCR system.

Discrimination between species can be achieved by

RE digestion of PCR products, which may generateRE fragments of different sizes which are unique to

meat and fat samples. M1-100 bp DNA ladder; 1, mutton; 2, beef; 3,

1 kb plus DNA ladder.

m meat and fat samples. M-100 bp DNA ladder; 1, mutton; 2, beef; 3,

1,12,13 and 14, lard; N-negative control (no DNA).

A.A. Aida et al. / Meat Science 69 (2005) 47–52 51

the animal type (Kocher et al., 1989; Meyer et al., 1995).

In this study, the PCR products from pork and lard

were digested with RE BsaJI, which generates the ex-

pected fragments of 131 and 228 bp (Fig. 3). Thus, the

standard restriction pattern for pork can be generated

by PCR-RFLP using pork meat.

4. Conclusion

A method suitable for routine extraction of genomic

DNA from meat and fat samples has been obtained with

the DNeasy� Tissue Kit (Qiagen). Using this kit, the ge-

nomic DNA was found to be suitable as PCR templates.This study shows that the mt-DNA is good enough for

routine detection of cyt b. In comparison of mt-DNA

between meats and fats, both mt-DNA are good enough

as PCR templates. These results indicate that PCR-

RFLP analysis of cyt b represents a powerful and easy

method for identification of species. It is a potentially re-

liable technique for detection of pig meat and fat from

other animals for Halal authentication.

Acknowledgements

The authors thank the Universiti Putra Malaysia

(UPM) for providing the funding (IRPA No. 03-03-

04-0172 EA 001) for this study.

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