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

Content

IntroductionObjectivesLiterature reviewMaterialsMethodsResultsDiscussionConclusion and recommendation

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.

IntroductionDNA-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.

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.

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.

Literature review

Food authenticationBecomes 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.

Literature review

Mitochondria cyt bAll 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.

Literature review

Agarose Gel electrophoresisProtocol 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

Literature review

PCR-RFLPSuitable 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.

Literature review

Agilent 2100 BioanalyzerA 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.

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.

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 37oc for Alu I

and Rsa I, and 60oc for BsaJ I.

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.

Methods

Detection of PCR-RFLP ProductsAgilent 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.

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

21B6

5 7

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

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

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

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

1000bpB7

6

5

4

3

2

1

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.

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.

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.

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).

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.

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 temperatureHas been keep in too long period of time.Has repeatedly of freeze and thaw process

which cause the DNA degradation.

Conclusion and recommendationPCR-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.

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.

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.

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.

THANK YOU