This document describes a study that aimed to identify chicken species in processed food products using PCR-RFLP analysis of the mitochondrial cytochrome b gene. The study collected 7 food samples, extracted DNA from them, amplified the cyt b gene using PCR, and digested the amplified products with restriction enzymes. The digestion products were analyzed via agarose gel electrophoresis and Agilent bioanalyzer to determine if patterns matched chicken DNA. The results showed PCR-RFLP analysis of the cyt b gene can authenticate chicken species in processed foods. Improved DNA extraction and testing from the start was recommended to obtain more accurate results.

1. IDENTIFICATION AUTHENTICATION OF
CHICKEN SPECIES FROM
PROCESSED FOOD PRODUCT BY
PCR-RFLP OF MITOCHONDRIAL CYT B
GENE.
FATIN HAKIMAH BTE RAMLI
2007128569
HS 221-08
SUPERVISOR:
PN. MAIMUNAH BTE MUSTAKIM

2. Content
Introduction
Objectives
Literature review
Materials
Methods
Results
Discussion
Conclusion and recommendation

3. Introduction
To select a suitable and safe food for our health,
food authenticity are important issues in labeling
process especially in processed food.
Authenticity identification of processed food
product is the most major concern in many
countries including Malaysia.
 A reliable and rapid method to identify animal
species in food and also an accurate method for
differentiation of meat species is very important.

4. Introduction
DNA-based identification methods have been
developed.
PCR-RFLP (Restriction Fragment Length
Polymorphism) is a method which is an
amplified fragment is cut by endonucleases by
recognized specific restriction sites into smaller
fragment (s) of different sizes.
This method is simple, robust, and much easier
to perform, inexpensive and can be used for the
identification of multiple species.

5. Objectives
General objective:
 The aim of this study is to
identify authenticity of chicken in
processed food samples by using PCR-
RFLP analysis of cyt b gene.

6. Objectives
Specific objectives:
 To determine authenticity of processed
chicken products using PCR-RFLP of cyt
b gene.
 To detect contamination of porcine in the
processed food samples by using
porcine cyt b gene.

7. Literature review
Food authentication
Becomes a major issue for food authorities.
Important to give an accurate information about the
product to consumer by improve the labeling
process.
The halalness of a product are important aspect of
the Muslim.
Species identification using PCR-RFLP of a
mitochondrial cytochrome b segment are the most
specific and sensitive techniques for food
components authentication.

8. Literature review
Mitochondria cyt b
All animal mitochondrial genomes contain the same 37
genes typically ~16 kb in size.
A partial DNA sequence of cytochrome b gene (cyt b)
can used to identify species origin of meat in processed
food.
The cytochrome b gene of mtDNA is a powerful
indicator for identify the species because it is more
sensitive.
The identification of the species can be based on
mutation in amplification products.

9. Literature review
Agarose Gel electrophoresis
Protocol that can be used to separate and
purify DNA and RNA fragments by apply an
electricity.
Separate the nucleic acid molecules by using
charge = DNA will move to the positive charge.
It is a simple and highly effective method

10. Literature review
PCR-RFLP
Suitable tool for tracing the meat origin in processed
food.
Involves amplification of DNA fragment followed its
digestion with appropriate selected restriction enzyme.
Enzyme will cleaves possible DNA strand, the
fragments are separate and can be detected by using
agarose gel electrophoresis.
Less costly for a routine food traceability analysis, rapid,
simple, robust and much easier to perform.

11. Literature review
Agilent 2100 Bioanalyzer
A unique analysis tool capable of handling nucleic
acids, proteins and cells on one platform.
Including with the chip kits can be help to speed up the
entire process.
Have high sensitivity, improved sizing accuracy and
automated, compare with regular gel electrophoresis.
Interpretation of data and handling specimen is
standardized.

12. Materials
Sample collection:
7 samples - fresh chicken, chicken stick, chicken hot
dog, chicken ball, fried chicken, chicken burger and
chicken nugget
Instruments and equipments:
BIO-RAD PCR MyCycler, Electrophoresis set, BIO-RAD
Gel-Doc, Agilent 2100 bioanalyzer, Micropipettes,
Microcentrifuge tube & Agilent DNA Chip.

13. Methods
 Samples Preparation
 PCR Products Amplification
 DNA Polymerase kit (FINNZYM DyNAzyme™ II)
 Done in BIO-RAD PCR MyCycler
 5 conditions – initial denaturation, denaturation, annealing,
extension & final extension.
 RFLP of Amplified PCR Products
 3 types of RE (restriction enzyme) - Alu I, Rsa I and BsaJ I.
 Digestion mixture was incubate for 1 hour at 37o
c for Alu I
and Rsa I, and 60o
c for BsaJ I.

14. Methods
Agarose gel preparation
2% agarose gel concentrations were used in
this study.
Detection of Restriction Enzyme Digestion
Enzyme digestion was detected by using
agarose gel electrophoresis.
Fragment size can be determined by using
BIO-RAD Gel-Doc analyzer.

15. Methods
Detection of PCR-RFLP Products
Agilent DNA 1000 kit
The sample, gel dye and ladder were load to the chip.
Put chip horizontally in the adapter and vortex for 1
minute at 2400 rpm.
Run the chip in the Agilent 2100 bioanalyzer within 5
minutes for obtain the result of DNA fragments.

16. Results and discussion
 DNA amplification of universal primer against (A) positive control,
(1-7) fresh chicken, chicken stick, chicken hot dog, chicken ball,
fried chicken, chicken burger, chicken nugget and (B) negative
control.
1000bp
600bp
400bp
50bp
A 4
3
21
B6
5 7

17.  DNA amplification of porcine primer against (A) positive control, (1-
7) fresh chicken, chicken stick, chicken hot dog, chicken ball, fried
chicken, chicken burger, chicken nugget and (B) negative control.
A B7
6
5
4
3
2
1
1000bp
300bp
50bp

18.  PCR-RFLP digestion of universal primer in Alu I restriction enzyme against
(A) positive control, (1-7) fresh chicken, chicken stick, chicken hot dog,
chicken ball, fried chicken, chicken burger, chicken nugget and (B) negative
control
1000bp
300bp
250bp
50bp
A 1
2
3
4
5
6
7
B

19.  PCR-RFLP digestion of universal primer in Rsa I restriction enzyme
against (A) positive control, (1-7) fresh chicken, chicken stick,
chicken hot dog, chicken ball, fried chicken, chicken burger, chicken
nugget and (B) negative control.
1000bp
400bp
50bp
150bp
250bp
500bp
A
1
2
3
4
5
6
7 B

20.  PCR-RFLP digestion of universal primer in BsaJ I restriction
enzyme against (A) positive control, (1-7) fresh chicken, chicken
stick, chicken hot dog, chicken ball, fried chicken, chicken burger,
chicken nugget and (B) negative control.
A
50bp
300bp
400bp
600bp
1000bp
B7
6
5
4
3
2
1

21.  PCR-RFLP digestion of universal primer and after digestion with Alu
I, Rsa I and BsaJ I restriction enzyme against fresh chicken, chicken
stick and chicken hot dog samples using Agilent DNA 1000 kit.

22.  PCR-RFLP digestion of universal primer and after digestion with Alu
I, Rsa I and BsaJ I restriction enzyme against chicken ball, fried
chicken and chicken burger samples by using Agilent DNA 1000 kit.

23.  PCR-RFLP digestion of universal primer and after digestion with Alu
I, Rsa I and BsaJ I restriction enzyme against chicken nugget
sample by using Agilent DNA 1000 kit.

24. Discussion
The PCR perform by using universal primer that
is cyt B1 and B2 to amplify the mitochondria cyt
b gene and also porcine primer that is F2 and
R1 to amplify porcine gene in the samples.
Samples were digested by using 3 different
types of restriction enzyme which is Alu I, Rsa I
and BsaJ I.
From E.coli strain that is Alu I (Arthrobacter
luteus), Rsa I (Rhodopsedomonas sphaeroides)
and BsaJ I (Bacillus stearothermophilus).

25. In PCR-RFLP analysis, the results will produce
by the computer.
The result is quite different from gel
electrophoresis which is each sample show
different readings and some sample was non
available (NA).
All of the other sample also most equal with
estimated result in gel electrophoresis.

26. Sample which did not show any result
may be caused by degradation of DNA.
Due to the
Techniques of storage.
Influence by the storage temperature.
Improper temperature
Has been keep in too long period of time.
Has repeatedly of freeze and thaw process
which cause the DNA degradation.

27. Conclusion and recommendation
PCR-RFLP method commonly used for
identification of food components.
Agilent DNA 1000 kit and Agilent 2100
Bioanalyzer are more sensitive and more
accurate to detect the species because the
result can be interpret directly from the
computer.
Authentication of chicken species in processed
food product can be identify by using PCR-RFLP
of mitochondria cyt b gene.

28. Conclusion and recommendation
To improve the study in identification of chicken
species in the processed food, this PCR-RFLP
method is suitable.
However, the sample collection and sample
preparation should be done again by ensure
appropriate storage condition.
Test must be done from beginning to get more
accurate result.

29. References
 Chandrika. M., Maimunah. M., Zainon. M. N., & Son. R. (2010).
Identification of The Species Origin of Commercial Available
Processed Food Products By Mitochondrial DNA Analysis.
International Food Research Journal, 17: 867-876.
 Chandrika, M., Zainon, M.N., Maimunah, M., Lesley, M.B., Jinap. S.,
& Son. R. (2009). Meat Species Identification and Halal
Authentication Analysis Using Mitochondrial DNA. Meat Science,
83:57-61.
 Daniel V. (2001). Agarose Gel Electrophoresis. Current Protocols in
Molecular Biology. 2.5 A.1– A.9.
 Erwanto. Y., Abidin. M. Z., Rohman & Sismindari. (2009). Pig
Species Identification In Meatballs Using Polymerase Chain
Reaction Restriction Fragment Lengtyh Polymorphism. The 1st
International Seminar on Animal Industry 2009.
 Fabrice, T. (2009). Molcular Identification Methods of Fish Species:
Reassessment and Posible Aplications. Rev Fish Biol Fisheries, 19:
265-293.

30. Ackowldgement
Most profound thanks to my supervisor, Puan Maimunah
Bte Mustakim for her supervision, guidance and all her
sacrifices during the on-going process of this project.
Thank to Dr. Roslinah Bte Mohammad Hussain as the
Head Programmed of BSc. (Hons.) Medical Laboratory
Technology for all her assistance and supports.
Thank to all lab staffs for their assistance, cooperation
and guidance during the process of this project in the
laboratory.

31. THANK YOU