LWT - Food Science and Technology 63 (2015) 714e719

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Sensitivity of polymerase chain reaction (PCR)-southern hybridization

and conventional PCR analysis for Halal authentication of gelatin
capsules
Sahilah Abd Mutalib*, Nursheila Mustafa Muin, Aminah Abdullah, Osman Hassan,
Wan Aida Wan Mustapha, Norrakiah Abdullah Sani, Mohd Yusof Maskat
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 12 March 2014
Received in revised form
23 February 2015
Accepted 4 March 2015
Available online 13 March 2015

Halal authentication of gelatin capsules was conducted using polymerase chain reaction (PCR)-southern
hybridization on chip and conventional PCR analysis. The primers used in PCR-southern hybridization
were targeted on mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene and its amplicon was 276 bp.
In conventional PCR, three pairs of mtDNA primers targeted cyt b, cytochrome oxidase II (COII) and ATP6
gene were tested, resulting of 398, 212 and 83 bp amplicons, respectively. Of 20 brands examined using
PCR-southern hybridization, 6 capsules (C1eC6) were found to be porcine DNA positive but none were
positive using conventional PCR method. The sensitivity of each primer in the detection of porcine DNA
was 0.25 ng (cyt b), 0.1 ng (COII), 0.001 ng (Olipro Chip) and 0.0001 ng (ATP6). Results demonstrated
that the PCR-southern hybridization on chip was useful and reliable for verifying porcine DNA in gelatin
capsules compared to conventional PCR.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Halal authentication
PCR-southern hybridization
Gelatin capsules
Conventional PCR
Porcine DNA detection

1. Introduction
Halal (permissible) quality of food is required to abide in Muslims daily live. However, the industrialization of food processing in
the 20th and 21st centuries has exposed Muslims community to
various ingredients such as gelatin in pharmaceutical product
arising from the advancement of science and technology. As a result
in many cases, the Muslims are facing difculties to ascertain which
products are permitted or not under the Islamic law. It is also inuence the other communities such as Jews, allergic toward
porcine ingredient and vegetarian. Thus, this study is the step of
preventing misconduct of pharmaceuticals products for consumer's
choice.
Gelatin is considered as a hydrocolloid which are special and
unique, serving multiple functions with a wide range of applications in various industries including beverages, processed foods,
cosmetics and pharmaceutical products (Karim & Bhat, 2008). The
use of gelatin in pharmaceutical products such as capsules is

* Corresponding author. Tel.: 60 03 89215444; fax: 60 03 89215410.

E-mail address: sahilah@ukm.edu.my (S.A. Mutalib).
http://dx.doi.org/10.1016/j.lwt.2015.03.006
0023-6438/ 2015 Elsevier Ltd. All rights reserved.

inevitable because it helps to protect the medicines against harmful

inuences, such as light and oxygen. There are two types of capsules namely soft and hard capsules. Both are different due to shell
composition and the production process. Soft gelatin capsules have
thicker shells and contain approximately 20%e30% of plasticizers in
the form of glycerol and sorbitol. High content of plasticizer will
hold approximately 30% of water content. While, in hard gelatin
capsules consist of pure gelatin with a sorption water content of
13%e16% (Stegemann & Bornem, 2002). In term of the production
process, hard gelatin capsules require less stringent conditions
during manufacture and storage than soft gelatin capsules. Details
on the production process of soft and hard gelatin capsules are well
explained by Stegemann and Bornem (2002). The soft capsules are
mainly used for liquid llings, while hard capsules are used for
powders.
The issue of gelatin is alarming and sometimes controversial due
to commercial gelatines are limited to porcine-based since the
emergences of Bovine Spongiform Encephalopathy (BSE) or mad
cow disease in the 1980s which restricted the use of bovine gelatin
(Morrison, Clark, Chen, Talashek, & Sworn, 1999). In addition the
production of porcine gelatin takes about 30 days, while bovine
gelatin production lasted between 60 and 80 days and this affect

S.A. Mutalib et al. / LWT - Food Science and Technology 63 (2015) 714e719

the product cost. The Halal gelatin from bovine source must fulll
the Shariah requirement such that the bovine must be slaughtered
by a Muslim and processed according to Shariah Laws. The alternative gelatin sources are gum arabic, seaweeds (carrageenan) and
shes (sh gelatin) but these could not fulll various industries
demand. The various use of porcine gelatin in industries is
expanding and the exposure of haram (non-Halal) gelatin is not
only towards Muslims but also other communities such as Jews,
vegetarians and a number of people who are allergic toward hidden
porcine ingredients and meat sources in processed foods (Tanabe,
Hase, et al., 2007).
There are a number of molecular techniques that can be used to
determine the presence of porcine DNA in food materials and to
identify disputability of the food products (Aida, Che Man, Wong,
Raha, & Son, 2005;Matsunaga et al., 1999; Montiel-Sosa et al.,
2000; Yoshida et al., 2009) and in feeds (Cheng, Wen, Ding, Koa, &
Kuo, 2003; Corona, Lleonard, Carpio, Uffo, & Martinez, 2007; Partis
et al., 2000; Tartaglia et al., 1998; Yoshida et al., 2009). Most early
techniques were based on hybridization to specic probes which is
time consuming (Chikuni, Ozutsumi, Koishikawa, & Kato, 1990;
Ebbehoj & Thomsen, 1991). However, the PCR techniques using
DNA amplication of specic target gene of mitochondria DNA
(mtDNA) is the method of choice due to its rapidity, specicity,
sensitivity and reproducibility (Tanabe, Miyauchi, et al., 2007). The
combination techniques of PCR and southern hybridization were
reported by Sahilah et al. (2012) in Halal market surveillance of
gelatin capsules in pharmaceuticals market in Malaysia. The PCRsouthern hybridization on chip was developed to detect the presence of porcine DNA by hybridizing the denatured biotinylated
amplicons with specic probes immobilized onto membrane. The
biotin-labeled amplicons bind to streptavidin-alkaline phosphatase
and subsequently detected by the colorimetric substrate of nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/
BCIP). The colored signal was captured by Scanner System which
allows the species-specic identication. Other technique for
porcine DNA detection in gelatin capsules using real-time PCR was
reported by Yasemin, Pelin, and Hamide (2012). In addition to the
cyt b gene, the primer targets used in conventional PCR for
detecting porcine DNA include cyt oxidase II (COII), D-loop, 12S
rRNA, 16s DNA, ATP8 and ATP6 (Cheng et al., 2003; Corona et al.,
2007; Partis et al., 2000; Tartaglia et al., 1998; Yoshida et al.,
2009). Therefore, this study was designed to compare the sensitivity of PCR-southern hybridization and conventional PCR in
verifying the porcine DNA present in the gelatin capsules.
2. Materials and methods
2.1. Gelatin capsules and DNA extraction
All samples (pharmaceuticals and porcine canned meats) were
purchased from Selangor during the period of August to September
2011. Twenty gelatin capsules of different brands (n 20) from
local and international companies were examined and designated
as C1 to C20. While, the three (n 3) different brands of porcine

715

canned meats (P1eP3) were purchased from local supermarkets. A

commercial Pig Genomic DNA (Novagen, Germany) was used as
positive control.
The capsules DNA were extracted using QIAGEN DNeasy Blood
and Tissue Kit (Qiagen, USA) as instructed by the manufacturer.
Total of 20 capsules were minced and a total of 50e100 mg was
transferred to a 1.5 ml sterile microcentrifuge tubes. DNA was
extracted from the capsules using QIAGEN DNeasy Blood and Tissue
kit (Qiagen, USA) and eluted with 100 ml of AE buffer. DNA was
quantied using MaestroNano Spectrophotometer (Maestrogen,
USA) and stored at 20  C until further analysis. All DNA of gelatin
capsules and porcine cannned meats were extracted in triplicate
from each source.
2.2. Oligonucleotide primers
The oligonucleotide primers targeting mitochondria DNA
(mtDNA) regions cyt b, cytochrome oxsidase II (COII) and ATP6 were
used in conventional PCR assays (Table 1). All mtDNA primers were
synthesized and supplied from First Base Laboratories (Selangor,
MY). A commercial primer targeting mtDNA cyt b gene (cyt b biotinlabeled oligonucleotide primers) for PCR-southern hybridization on
chip (Olipro PORCINE Gene Chip Kit) was supplied by OLIPRO
Biotechnology Sdn. Bhd. MY.
2.3. PCR amplication using different oligonucleotide primers
Amplication of DNA using primers SimP F and SimP R targeting
the mtDNA cyt b at 398 bp (Matsunaga et al., 1999) were performed
in a nal volume of 50 ml containing 25 ml of DreamTaq PCR Master
Mix (2X) (Fermentas, Lithuania), 1 ml of 5 mM each primer (forward
and reverse for porcine DNA), NFW and 2 ml of approximately 50 ng
DNA template depending on the DNA concentration. A negative and
a positive DNA control was performed by adding 2 ml of NFW and
Pig Genomic DNA (Novagen, Germany) respectively. The condition
of porcine DNA amplication assay consisting of the initial denaturation at 95  C for 2 min, followed by 35 cycles of amplication at
94  C for 30 s (denaturation), hybridization at 55  C for 30 s, and
elongation at 72  C for 40 s, and a nal extension step at 72  C for
3 min. It was carried out in the Mastercycler gradient thermal
cycler (Eppendorf, USA). The amplication products were electrophoresed through 2.5% (w/v) agarose gel in 1 X TAE buffer (40 mM
Tris-OH, 20 mM acetic acid and 1 mM of EDTA; pH 7.6) at 100 V for
45 min and stained in ethidium bromide.
The PCR amplication using primers pork 1 and pork 2 for COII
at 212 bp size (Lahiff et al., 2001) were performed in a nal volume
of 50 ml containing 25 ml of DreamTaq PCR Master Mix (2X) (Fermentas, Lithuania) 1 ml of 100 mM each primers (forward and
reverse), NFW and 2 ml of approximately 50 ng DNA template. A
negative and a positive DNA control were performed as above.
Mastercycler gradient thermal cycler (Eppendorf, USA) was used
to run the PCR with a temperature program consisting of the initial
denaturation at 95  C for 2 min, followed by 30 cycles of 94  C for
1 min, 55  C for 1 min, 72  C for 2 min, and a nal extension step at

Table 1
Oligonucleotide primers for porcine DNA detection in gelatin capsules.
Primer sequence (50 to 30 )

Gene target

Amplicon length (bp)

Reference

SimP-F: 50 -GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA-30
SimP-R: 50 -GCT GAT AGT AGA TTT GTG ATG ACC GTA-30
Pork 1: 50 -GCC TAA ATC TCC CCT CAA TGG TA-30
Pork 2: 50 -ATG AAA GAG GCA AAT AGA TTT TCG-30
PPA6 F: 50 -CTA CCT ATT GTC ACC TTA GTT-30
PPA6 R: 50 -GAG ATT GTG CGG TTA TTA ATG-30

Cytochrome b

398

Matsunaga et al. (1999)

Cytochrome oxidase II

212

Lahiff et al. (2001)

ATP6

83

Yoshida et al. (2009)

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S.A. Mutalib et al. / LWT - Food Science and Technology 63 (2015) 714e719

72  C for 10 min. The PCR products were analyzed through 2.5% (w/
v) agarose gel as mentioned above.
The porcine DNA were amplied using 83 bp target primers of
PPA6F and PPA6R (ATP6) (Tanabe, Miyauchi, et al., 2007) in a 20 ml
reaction volume containing 10 ml of DreamTaq PCR Master Mix (2X)
(Fermentas, Lithuania), 1 ml of 0.4 mM each primer, 6 ml of NFW and
2 ml of approximately 50 ng extracted DNA template. A negative and
a positive DNA control were performed as above. PCR was also
carried out in Mastercycler gradient thermal cycler (Eppendorf,
USA) with a temperature program consisting of the initial heat
activation at 95  C for 9 min, followed by 45 cycles of 92  C for 30 s,
55  C for 30 s, 72  C for 30 s, and a nal extension step at 72  C for
5 min. The PCR products were separated by electrophoresis through
3% (w/v) agarose gel in 1X TAE buffer (40 mM Tris-OH, 20 mM
acetic acid and 1 mM of EDTA; pH 7.6) at 80 V for 1 h and stained in
ethidium bromide.
All agarose gels in above experiments used 100 bp DNA ladder
(Fermentas, Lithuania) as the size marker and was visualized using
UV transilluminator gel documentation (AlphaImager EP System,
India).
2.4. PCR amplication for southern-hybridization on DNA chip
The southern-hybridization on Olipro PORCINE Gene Chip
was started with PCR amplication by using Olipro Porcine PCR
kit. The multiplex PCR is performed with biotin-labeled primer sets
to amplify species-specic target DNA (cyt b) and internal control
sequences. Later, the corresponding pair of sequence-specic
probes are immobilized onto membrane and hybridized with the
biotinylated PCR products. PCR was conducted as manufacturer's
instruction in a nal volume of 50 ml reaction. Each reaction
mixture containing 24.6 ml of Porcine Gene Chip 1 X PCR Master Mix
(Olipro, MY), 0.5 ml of Taq DNA Polymerase, 2.0 ml of approximately 50 ng DNA template, and nuclease free water (NFW) as mark
up to nal volume. A negative and a positive DNA control was
performed by adding NFW and Pig Genomic DNA (Novagen, Germany), respectively. The PCR amplication was carried out in
Mastercycler gradient thermal cycler (Eppendorf, USA) with a
temperature program consisting of the initial denaturation at 95  C
for 5 min to completely denatured the DNA template, followed by
45 cycles of denaturation at 95  C for 30 s, annealing at 55  C for
30 s, polymerization at 72  C for 30 s and nally, elongation at 72  C
for 5 min. Negative controls (NFW) were included in each PCR
amplication, in order to verify the PCR efciency and to detect
contamination (Sahilah, Norhayati, Norrakiah, Aminah, & Wan
Mustapha, 2011). The amplication products were analyzed by
electrophoresis using 2.5% (w/v) agarose gel in 1 X TAE buffer
(40 mM Tris-OH, 20 mM acetic acid and 1 mM of EDTA; pH 7.6) at
100 V for 45 min and stained in ethidium bromide and visualized
with UV transilluminator gel documentation (AlphaImager EP
System, India). A 100 bp DNA ladder (Fermentas, Lithuania) was
used as size reference. Positive result for porcine DNA was indicated
by a band of 276-bp, and the 195-bp internal control (IC). IC in each
PCR functioned as an indicator to ensure that all PCR assays are in
good condition or the reactions were carried without inhibitor or
impurities (personal communication).

hybridize the biotinylated amplicons with specic probes. After

hybridization reaction, the chips were again washed for color
development. Chips were rinsed using reagent G and oven-dried
(OliproOven HYB001, MY) at 70  C for 5 min. Finally, chips were
scanned and positive spots identied using Scanner System (OliproScan, MY). Positive result for porcine DNA will be indicated by
perfect matched probe-target hybrid which formed blue-purple
color on the chips.
2.6. Interpretation of results
Positive detection of porcine DNA is showed by the grey color at
two spots in the middle, while for internal control, eleven spots at
the left and the upper part of the chip appeared, including 4 spots
located at every corner of the chips. However, no color will be
formed in the middle of the chip if the result is negative. If the color
does not appeared at all internal control spots, results are not valid,
thus the experiment should be repeated.
2.7. Detection limit of oligonucleotide primers
The detection limit of all oligonucleotides as described in Table 1
was examined using Pig Genomic DNA (Novagen, Germany). The
PCR assay condition was similar as described in the PCR amplication using different oligonucleotide primers with different concentration of porcine DNA ranging from 0 to 150 ng. While, in PCRsouthern hybridization analysis, the condition used was similar as
described with porcine DNA but the concentration ranged from
0.00001 to 1 ng.
3. Results and discussion
In the presence study, we examined twenty gelatin capsules for
the presence of porcine DNA using PCR-southern hybridization on
chip. Of twenty samples tested, six capsule samples were tested
positive for porcine DNA (C1eC6). As indicated in Fig. 1, positive
results for porcine DNA showed by bands of 276 bp and the 195 bp
(IC). Capsules C1 and C3 did not show band of 276 bp on agarose gel
analysis, but gave strong signal when it hybridized on the chip
(Fig. 2). Porcine DNA positive showed two spots in the middle of the

2.5. Southern-hybridization analysis on chip

The amplicons were denatured at 95  C for 10 min and placed
into ice block immediately. Hybridization was carried out following
PORCINE Gene Chip protocols (Olipro, MY). The amplicons were
mixed with reagent A on the chips and incubated in hybridization
oven (OliproOven HYB001, MY) at 70  C for 1 h with maximum
vibration. Chips were washed using reagents B, C, D, E and F to

Fig. 1. Gel electrophoresis of gelatin capsules using primers supplied by Olipro

Porcine PCR kit before southern hybridization on gene chip analysis. Lane M; Marker
(100 bp ladder); Lane 1e6: Capsule samples of C1eC6 (Positive of porcine DNA
(276 bp) and internal control (195 bp)); Lane 7: Positive control and Lane 8: Negative
control.

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717

Fig. 2. Pattern images on porcine gene chip of capsule samples which was read and identied by scanner analysis software. Chip image results of positive porcine 2 spots at the
center for capsule samples (C1eC6) and porcine canned meats (P1eP3) on Olipro Porcine Gene Chip. The other eleven spots were internal control (IC). Chip image results of
positive control spot (PC) and negative control spot (NC) on Olipro Porcine Gene Chip.

chip (Fig. 2) with eleven spots as internal control (IC). All internal
control for capsule samples showed clear bands on agarose gel
which indicated the PCR assay was in a good condition. There were
two other spots which were invisible (negative control) and it position are located above the two positive spots. If the above two
spots were positive to porcine DNA, this indicated the chip was
contaminated with porcine DNA prior to used. Table 2 summarized
the porcine DNA detection on gelatin capsules and porcine canned
meats using PCR-southern hybridization and conventional PCR
analysis. The result obtained from this study was consistent with a
previous study (Sahilah et al., 2012) which reported similar
observation on Halal market surveillance of one hundred and
thirteen gelatin capsules from different pharmaceutical products

using PCR-southern hybridization on chip. In their nding, low

intensity band was observed on agarose gel may due to the most of
DNA was degraded and loss of specic site on mtDNA target region.
However, the formation of nitroblue substrate of NBT/BCIP (Fig. 2)
at low amplicons concentration, hybridized with specic probes on
chip membrane and give positive result. Those results were
consistent in repeated experiments. Other studies showed that
despite DNA being degraded and altered, it was possible to amplify
small DNA fragments with sufcient information to allow identication (Corona et al., 2007; Rodriguez et al., 2004; Teletchea,
Maudet, & Hanni, 2005).
The sensitivity of PCR-southern hybridization on DNA chip was
compared to the conventional PCR techniques as described by

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S.A. Mutalib et al. / LWT - Food Science and Technology 63 (2015) 714e719

Table 2
Summarization of porcine DNA detection on gelatin capsules and porcine canned meats using PCR-southern hybridization and conventional PCR analysis (analyzed on agarose
gels).
Samples

C1
C2
C3
C4
C5
C6
P1
P2
P3
C7eC20

Type of samples

Hard capsule
Soft capsule
Hard capsule
Hard capsule
Soft capsule
Soft capsule
Porcine canned meat
Porcine canned meat
Porcine canned meat
Hard and soft capsule

PCR-southern hybridization on DNA chip

All negative

Matsunaga et al. (1999). The amplicons of cyt b region gave a single

band of 398 bp in size. No band was observed for all capsules and
porcine canned meat (P1) in repeated experiments. However, two
samples of porcine canned meats (P2 and P3) were showed positive
band which produced a band of 398 bp in size (Table 2). Matsunaga
et al. (1999) reported these primers were useful to detect porcine
DNA in a range of fresh to cook meat (100e120  C for 30 min).
However, heat treatment applied to canned food in industrial
practices could possibly damage the mtDNA resulting in loss of
specic primer binding sites as indicated in canned meat sample of
P1 which not produced any band. This happened because there are
several of heat treatments involved during production processes of
canned food such as cooking, pasteurization, sterilization and so on
in the condition of high pressure where the temperature parameter
might be up to 100  C for 10e60 min and are exposed to a pH < 4
(Teletchea et al., 2005).

Conventional PCR
Cytochrome b (398 bp)

Cytochrome oxidase II (212 bp)

ATP6 (83 bp)









All negative








All negative








All negative

The COII and ATP6 primers were subsequently used to detect

porcine DNA in all gelatin capsules (C1eC20). Both primers were
chosen due to its capability to detect porcine DNA in feed samples
which undergone heat and denaturing treatment during
manufacturing. Similar assumption goes to gelatin capsules which
also undergone various steps of gelatin manufacturing process.
However, no specic band was observed for both primers in
detecting porcine DNA in gelatin capsule. From Lahiff et al. (2001)
and Yoshida et al. (2009), those primers were reported useful to
detect porcine DNA in feeds (Lahiff et al., 2001; Yoshida et al., 2009)
with amplicons of 212 bp and 83 bp in size, respectively. The
porcine DNA detection of PCR-southern hybridization and conventional PCR analysis of gelatin capsules were shown in Table 2. As
indicated in Table 2, the 6 capsules tested which were positive towards porcine DNA using PCR southern-hybridization analysis
showed negative results in PCR conventional analysis.

Fig. 3. Porcine gene chip pattern images of different porcine genomic DNA concentration. NC1 and NC2 were negative control; 1: 10 ng; 2: 1 ng; 3: 0.1 ng; 4: 0.01 ng; 5: 0.001 ng: 6:
0.0001 ng and 7: 0.00001 ng.

S.A. Mutalib et al. / LWT - Food Science and Technology 63 (2015) 714e719

The detection limit of the commercial Olipro PORCINE Gene

Chip primer (Olipro, MY) in the detection of porcine DNA based
on cyt b target sequence was 0.001 ng (1 pg) (Fig. 3). While, for the
other different primers were 0.25 ng (cyt b), 0.1 ng (COII) and
0.0001 ng (ATP6) (Lahiff et al., 2001; Matsunaga et al., 1999;
Yoshida et al., 2009). Our detection limit results are consistent
with the ndings reported from those other workers (Lahiff et al.,
2001; Matsunaga et al., 1999; Yoshida et al., 2009) who reported
the similar values of genomic DNA detection limit.
The detection limit of ATP6 (PPA6F and PPA6R) primers showed
high sensitivity to detect porcine DNA which the DNA was come
from the commercial source. However, those primers failed to show
any specic band on agarose gel from gelatin capsule samples. Our
nding was in contrast with other workers who reported the
primers were successful to detect porcine DNA in meat and bone
meal (MBM), thus, useful as an Ofcial Methods of Feed Analysis
(Matsunaga et al., 1999; Yoshida et al., 2009). They demonstrated
the ATP6 primers have high specicity and sensitivity in detecting
porcine DNA in various rendering procedures in Japan. The possible
reason why it failed to detect porcine DNA from gelatin capsule was
the mtDNA not only degraded but also present in small quantities
which reduced the number of DNA fragments with suitable size for
molecular analysis (Teletchea et al., 2005). The respective amplicon
may exist in a very low concentration and could not be seen on
agarose gel analysis.
4. Conclusions
In conclusion, the PCR-southern hybridization on chip is a new
innovative approach in Halal authentication, though combination
of PCR and southern hybridization are not new techniques. The
advantage of this technique is that, they are able to detect a very
low amount of porcine DNA amplicons after PCR process by
southern hybridization procedures. The PCR-southern hybridization offer promising results in detecting porcine DNA in highly
processed products such as gelatin and other processed foods. This
approach demonstrated sensitive, reproducible and certainly useful
to detect porcine DNA for Halal authentication which would further
validate the pharmaceutical products labeling for consumers.
Acknowledgments
We are gratefully thanks to Olipro Biotechnology Sdn. Bhd.,
HEJIM grant (INDUSTRI-2011-052) and FRGS/1/2014/STWN10/
UKM/02/4, for the assistance in this research.
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