SEMINAR REPORT

BTE 3191
Molecular Approach in Authentication of Halal
Food

Muhammad Arif bin Zuhairi 1220775

Muhammad Nor Wazien bin Norrashid 1222807
Supervisor: Prof. Dr. Irwandi Jaswir
Coordinator: Dr Fazia Adyani binti Ahmad Fuad

Content
Introduction
Halal and Haram issues
Electrophoresis
Approach
PCR Approach
LAMP Approach
Conclusion
Discussion

Pag
e
1
1
3
5
9
12
12

Introduction
Food is an important aspect for human. Basically it provide us energy
for daily works. As modernity is going forward, people comes with variety of
food productions to meet the demands and desires, to serve meals with the
best diet quality, to promote to others the new discover of food recipes, and
to make people happy enjoying the beautiful of taste.
For Muslims, special consideration of Halal status need to be followed
as a compulsory guideline for food consumption. The establishment of Halal
status by Allah S.W.T. is a great gift and a proof of Allahs mercy to His
creatures. The benefits of consuming Halal food cover the physical,
spiritual, emotional and intellectual aspects. In Quran, one verse that
mentions about Halal is from the second verse of Surah Al-Baqarah, O
mankind, eat from whatever is on earth (that is) lawful and good and do not
follow the footsteps of Syaitan. Indeed, he is to you a clear enemy. There
are many other verses mentioned by Allah S.W.T. in Quran about Halal
issue.
Also the restriction of taking Haram food, inverse of Halal is
supported by a hadith narrated by Sayyidina Abu Bakar RA. From him,
Rasulullah SAW said that That body will not enter Paradise which has been
nourished with Haram. Hadith by Baihaqi. Thus for Muslim, Halal and
Haram concept is a crucial consideration to be observed and well
established in life to achieve the ultimate pleasure of Allah S.W.T.

Halal and Haram issues

Surely there are many issues regarding Halal and Haram of the food
made and manufactured outside there. In Malaysia, Department of Islamic
Development Malaysia (JAKIM) shares statements about some issues on the
website, which related to Halal and Haram food.
Some of the issues are the production of new types of food, animal
based and imported food ingredients, slaughtering method, food additives,
facilities, Genetic Modified Organism, food-drug interface, new hazard
substances and adulteration of food.
In 2014, there was an interesting issue about Cadbury Chocolate which
was expected to contain DNA of pig. This shocking news had urge some
related parties to investigate for the truth. It shows how important for
Muslims to be alert on this issue and it could be a new platform for
responsible parties to implement ideas, modules and approaches in order to
cope this issue.
1

In the perspective of science, there are some possible and practical

ways to detect non-halal constituents in the food sample. Scientists keep
working on finding efficient approaches to trace non-halal ingredient in order
to keep food in the markets are valid for Muslim according to Shariah
compliance.

Some techniques established and proposed by scientists are:

1. Microscopic method.
2. Lipid-based method.
- Analysis of volatile organic compound.
- Gas chromatography-mass Spectrometry solid-phase
microextraction.
- Electronic nose.
3. Protein-based method.
- Quantification of Histidine Dipeptides.
- Analysis of Species-Specific Osteocalcin Analysis of Muscle
Proteins.
- Detection of Species-Specific Proteins by ELISA.
4. DNA-based method.
- Species-Specific PCR (SSP).
- Temperature/Denatured Gradient Gel Electrophoresis.
- PCR-Restriction Fragment Length Polymorphism.
- DNA Barcoding.
- Real-time PCR.
- Oligonucleotide Microarray.
5. Nanotechnology in DNA Detection.
- Species Detection Using Gold Nanoparticles.
6. Future Technology to Detect Species in Foods and Feeds.
- Nanogap Electrodes for the Label Free Detection of DNA.
- Planar Nanogap Devices for DNA Detection.
- Nanogap Detectors in Nanofluidics.
- MOSFET and ISFET.
- NanoMIM Devices for Sensing DNA Target.
- Carbon Nanotube-Based Nanogap Devices for Sensing DNA.
- Quantum Dots in Label-Free DNA Sensing.
- Surface Enhanced Raman Spectroscopy for Label Free DNA
Detection.
2

In accordance to this project, the molecular approaches we interested

with are PCR, electrophoresis and LAMP. For the definitional purpose, the
term molecular refers to operation involving cell, DNA, RNA, protein and
other very small and basic elements. Authentication is a phrase related to
identification and confirmation.

Electrophoresis approach
Case Study Journal: Identification polypeptide biomarkers of porcine skin gelatin
by two-dimensional electrophoresis.
General steps:
i. Prepare the gel.
ii.
Prepare the sample.
iii.
Prepare the buffer.
iv.
Choose molecular weight buffer.
v. Run the gel.
vi.
Stain the gel.
vii.
Post staining.
Introduction
Gelatin is widely used in food products and medicinal items commonly found
in yogurt, ice cream, desserts and sweets. It has high molecular weight, a
polypeptide derived from collagen which is obtained by partial hydrolysis of
the collagen that present in bones and hides, mainly from bovine or skin
from pig. Consists of two types A and B based on acid-treated or alkalitreated processes. Two-dimensional electrophoresis (2-DE) is used as a
powerful separation technique. This separation of gelatin polypeptides
mixture is basically according to its molecular weight and isoelectric point.
PDQuest software also used together with 2-DE to identify prominent spots.
Materials
Porcine skin gelatin was purchased from 3 different companies, bovine skin
gelatin, ready immobiline pH gradient strip, urea, 1.5M Tris HCL buffer pH 8,
CHAPS, acetone, Iodoacetamide FAST-Silver Staining kit, Thiourea and
bromophenol blue, Glycerol, Ethanol, and Glacial acetic acid.
Methods
3

Preparation of samples, isoelectric focusing (IEF), Sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE), Image analysis and
Adulteeration.

Result and discussion

Potential biomarker of porcine skin gelatins were obtained. The porcine skin
gelatins for all three different companies had pI ranged from pH 5 8. Some
difficulties due to presence of salts, minerals, and other compound. 2-DE has
high resolution, powerful separation of spots proteins and high
reproducibility. PDQuest software identified 10 dominant proteins spots. The
detection of porcine gelatin will be using these 10 selected spots as
biomarkers. For adulteration test, pure and adulterated samples were
compared to see the presence of 10 selected protein spots decided earlier.
PDQuest software is used to make landmark on some of the dominant spots
in the gel to assist differentiating the protein spots. They were using pixel
intensity to measure the intensity of the protein spots. A graph Intensity
versus Spot was made to see the presence of any 10 protein biomarkers in
5to 1% adulteration of pig skin gelatin. The possible reason for the presence
and absentee of protein spots at certain level of adulteration is due to
variation of 2-DE gel experiment that affect the measured protein levels.
Normalization step had been performed to minimize the experimental
variations.

Conclusion
10 biomarkers gelatin polypeptides of porcine skin were successfully
identified by 2-DE. High resolution of proteins separation in 2-DE gel can be
obtained by extraction of protein from gelatin using buffer followed by
protein precipitation using cold acetone. 2-DE method has the ability to
detect as low as 1% level of adulteration in gelatin samples. The data
represented can serve as basis in identifying the sources of gelatin for
authentication purposes.

PCR Approach
PCR stand for polymerase chain reaction. It is most widely use technique in the biotechnology
field for many purpose especially in DNA analysis like food processing. As the name indicate the
technique use polymerase enzyme to add nucleotide and create a chain reaction or basically
amplify template DNA to create a copy segment of it repeatedly.
The fundamental of polymerase chain reaction consist of 3 step. Firstly is the denaturing step.
The DNA is heated at its melting temperature usually at 90C for about 1 minute to 2 minute so
that the double strand DNA will split apart. Next is the annealing step. In this step temperature is
dropped to 50C to 60C allowing the primer to bind to the complementary strand of the
template DNA. Finally is the extension step. The temperature in this step is increased to 70C for
1 minute to 2 minute this allow the thermostable polymerase to elongate the new complementary
stand from the template DNA which start at the primer where our target sequence is identify
before the PCR process.
Some of the key element that has to be taken when doing the PCR is melting DNA temperature.
Although the denature temperature are usually 90C, different species or organism has different
temperature for the DNA to denature. It is also important to design the correct primer which can
bind to the complementary strand of DNA near the target sequence. DNA polymerase is also the
key player in manufacturing new DNA copies usually Taq polymerase are used which was
isolated from bacteria living in hot spring called Thermus aquaticus.

There are actually different variation of PCR for example colony PCR, nested PCR, Multiplex
PCR, Inverse PCR, long PCR, Reverse Transcriptase PCR, Real time PCR and many more.
Real Time PCR
Real time PCR will be elaborate in detail since one of our case study of molecular analysis on
halal food uses this method. As the name indicate this PCR technique is similar to the
conventional PCR method the only different is that we are able to monitor the amplification of
template DNA duplicated in real time not at the end. It is actually monitor by the emission of
fluorescence during each cycle. The thermocycle which denature, anneal, and elongate the DNA
template basically is connected to the spectrofluorometer which detect the intensity of the
fluorescence. Fluorescence bind to the DNA so as the number of gene increase the number of
fluorescence will also increase. Usually the increase of fluorescence is measured during the
exponential phase of PCR by quantification.
There are generally two type of probe for the detection of fluorescence which are non-specific
detection and specific detection. Some example of non-specific detection are SYBR Green I dye
and example of specific detection is TaqMan.

SYBR green is a DNA binding dye which can only bind to a double strand DNA at the minor
groove segment. The dye will emit light upon binding. In other word the more the double strand
DNA the more binding will occur and more fluorescence will be detected. So it is a perfect
mechanism for analysis and forensic purpose. Some of the main advantage of this method is that
it is relatively cheap, doesnt require probe design unfortunately it is not specific which can lead
to the false positive of fluorescence, for example it can bind to the primer dimer since it is in
double strand.
SYBR green dye binding during each PCR phase: 1(denature), 2(anneal), and 3(elongate):

TaqMan is a dye which consist of two part the quencher dye and the reporter dye. The reporter
dye have high tendency to emit photons but since it is distributed freely it is always near the
quencher dye which block the reporter from emitting photons. During anneal process the dye
will attach to the DNA strand as the Taq Polymerase elongate the DNA it will cut of the reporter
dye from the quencher dye. Freeing the reporter dye will make it emit photons and increase the
intensity of fluorescence. Some of the main advantage of this method is that it is very specific
hence it does not detect non-specific PCR product. Unfortunately it is more difficult to design,
sometime the TaqMan probes do not quench efficiently and it is more expensive.
TaqMan separate quencher dye from reporter dye so fluorescence can be emitted:

Case Study Journal: Analysis of Pork Adulteration in Commercial Burger Targeting PorcineSpecific Mitochondrial Cytochrome B Gene by TaqMan Probe Real-Time Polymerase Chain
Reaction
This journal explain about the use of species-specific primer and TaqMan probe to detect and
amplify 109bp of swine mt-cyb gene. Firstly sample was collected in triplicate in three different
days. Some of the sample collected was fresh raw muscle tissue of pig, cow, sheep, goat, deer,
and hen. Also five fish species cichlid, shad, shrimp, tuna and cuttlefish. Commercial burger
containing pork also purchase. Beef and chicken burger from McDonald and KFC was also
bought as sample.
Calibration of different percentage of pork was mixed with beef burger to see the fluorescence
signal response for 0.01, 0.1,1 and 10% contamination of pork meat in beef burger.
This test want to show how efficient and accurate is the technique in detecting pork
contamination in food. Two pair of primer use are SwcytbF and SwcytbR which basically target
the 109bp fragment of swine cytb gene. The TaqMan probe used was SwcytbTqM consisting of
6-FAM and 3-IABkFQ, an addition of ZEN probe was added as the quencher since the probe is
quite long this help reduce the background signal in amplicon detection.
Result of Fluorescent profile of PCR product amplify: A and D (from porcine specific) B is
animal and C is fish (from endogenous PCR system):

Result quantification cyle Cq of porcine PCR and endogenous PCR:

Result of fluorescence profile of PCR product obtain from beef burger with pork adulteration A
(100%), B (10%), C (1%), D (0.1%) and E (0.01%) and F (0%)

All this result verify how accurate is TaqMan probe technique in detecting swine cytb gene. Even
at contamination of 0.01% of pork the probe can detect it.

Loop Mediated Isothermal Amplification (LAMP) Approach

LAMP is also an amplification technique for isothermal nucleic acid. It differ from PCR
technique since it does not need the change in temperature for denature, anneal and elongate.
Instead the temperature remain constant about 60C to 65 C. The polymerase used are high
strand displacement activity and replication activity. The primer used identify 6 distinct region
on target gene so 4 different primers are design. This cause high specificity of target sequence.
The product of amplification can be detect with the naked eye because of the increase quantity of
pyrophosphate precipitate a by-product of amplification. So reaction can be monitor in real time
by fluorescence dye like SYBR green, the colour change can actually be seen with the naked eye
so no need for expensive equipment to confirm the product of target sequence.
Some advantages of LAMP is that it can amplify faster than PCR, it is also cheap and simple
process. Since the temperature is constant not much energy is used compare to normal PCR so
economically it is better. Unfortunately it is less versatile than PCR it scope is more for analysis
and detection not for cloning. To add to that the primer design is much harder and less freedom
to choose.
9

DNA is amplify using the LAMP technique notice loop is form at the end:

Case Study Journal: Development of a Rapid Method for the visible Detection of Pork DNA in
halal Products by Loop-Mediated Isothermal Amplification.
This journal use the method of LAMP to detect pork contamination, the technique visibly
identify pork DNA in meat product. 4 pig specific primer were designed according to the
mitochondrial DN1 gene sequence. The primer use are inner primer (FIP and BIP) and outer
primer (F3 and B3). Bst DNA polymerase was used and amplification of DNA was done under
isothermal temperature at 60 to 65 degree Celsius. As DNA is amplify pyrophosphate ion is
produced thus colour can be visible in presence of calcein and confirming pork contamination.
There are 4 tested carried out. Firstly LAMP technique was test on different meat which are
cattle, buffalo, sheep, goat, dog, rabbit, rat, chicken, duck, fish and pig. This test the specificity
of the technique. Secondly, pork detection was carried out on 50ng, 5ng, 0.5ng, 50pg, 5pg, 0.5pg,
50fg, 5fg, 0.5fg of pork sample to see how accurate the detection is. Thirdly, pork is mix with
beef with 0%, 0.01%, 0.1%, 1%, 10% and 50% of contamination. Lastly, the pork was heated in
different way in water at 80C for 30 minute and 60 minute, in water at 100C for 30 minute and
60 minute, autoclave at 121C for 30 minute and microwave heated. This is to see whether DNA
will denature and disintegrate and can LAMP detect it.
10

Lane 1 to 13 (pig, cattle, buffalo, sheep, goat, fox, dog, rabbit, rat, chicken, duck, fish and no
template control):

Lane 1 to 9 pork sample (50ng, 5ng, 0.5ng, 50pg, 5pg, 0.5pg, 50fg, 5fg, 0.5fg and NTC):

Lane 1 to 6 pork mixed with beef (50%, 10%, 1%, 0.1%, 0.01%, 0% and NTC):

Lane 1 to 8 heating process of pork(water at 80C for 30 minute, water at 80C for 60 minute,
water at 100C for 30 minute, water at 100C for 60 minute, autoclaved at 121C for 30 minute,
fried for 20 minute, microwave for 20 minute, raw and NTC.)

The result clearly show how effective is LAMP method in detecting pork adulteration. It is
specific since it only detect pork sample, the accuracy is up to 0.5pg sample, it detect all the
mixture of pork with beef even at only 0.01% contamination and finally detect all the pork
undergone different heating method which is no surprise since DNA is a stable substance.

11

Conclusion
We can conclude that all this technique molecular are reliable in detecting pork adulteration in
food. The main thing in analysis is the rate of contamination detection by this method of course
real time PCR and LAMP triumph over electrophoresis. Real time PCR is more complicated than
LAMP in term of cost and energy usage because of the process while LAMP is quite simple
because the detection can be seen with the naked eye and not much cost and energy needed to
perform the analysis. Although in term of functionality PCR is more diverse but for quick
analysis to get result LAMP is definitely better
Quick, efficient, accurate and cheap is our key in choosing the best analysis method which is
LAMP method.
Reference

Ali, M., Hashim, U., Dhahi, T., Mustafa, S., Man, Y. and Latif, M. (2011). Analysis of
Pork Adulteration in Commercial Burgers Targeting Porcine-Specific Mitochondrial
Cytochrome B Gene by TaqMan Probe Real-Time Polymerase Chain Reaction. Food
Analytical Methods, 5(4), pp.784-794.

12

Huang, Q., Xu, T., Wang, G., Huang, J., Xia, H., Yin, R., Tang, A. and Fu, W. (2011).
Species-specific identification of ruminant components contaminating industrial crude
porcine heparin using real-time fluorescent qualitative and quantitative PCR. Anal
Bioanal Chem, 402(4), pp.1625-1634.
Ran, G., Ren, L., Han, X., Liu, X., Li, Z., Pang, D., Ouyang, H. and Tang, X. (2015).
Development of a Rapid Method for the Visible Detection of Pork DNA in Halal
Products by Loop-Mediated Isothermal Amplification. Food Analytical Methods.

Aina, M.A., *Amin, I., Raja Mohd Hafidz, R.N. and Yaakob, C.M. (2013). Identification
polypeptide biomarkers of porcine skin gelatin by two-dimensional electrophoresis.
International Food Research Journal 20(3), pp.1395-1399
Clark, D. (2005). Molecular biology. Amsterdam: Elsevier Academic Press.
Ali, M., Kashif, M., Uddin, K., Hashim, U., Mustafa, S. and Che Man, Y. (2012). Species
Authentication Methods in Foods and Feeds: the Present, Past, and Future of Halal
Forensics. Food Analytical Methods, 5(5), pp.935-955.
Hamzah, A. (2014). Porcine DNA Detection in Finished Meat Products Using Different
Mitochondrial DNA (mt-DNA) on Polymerase Chain reaction. J Nutr Food Sci, 04(06).
Nicholl, D.S.T. (2008) An Introduction to Genetic Engineering, 3rd Edition, Cambridge
University Press
Nhari, R., Ismail, A. and Che Man, Y. (2012). Analytical Methods for Gelatin
Differentiation from Bovine and Porcine Origins and Food Products. Journal of Food
Science, 77(1), pp.R42-R46.

Sandra Hermanto*, La Ode Sumarlin, Widya Fatimah. (2013). Differentiation of Bovine

and Porcine Gelatin Based on Spectroscopic and Electrophoretic Analysis.J.Food
Pharm.Sci, 1, pp.68-73.

13