Fatin fyp ppt sbnr

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Fatin fyp ppt sbnr

  1. 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. 2. Content Introduction Objectives Literature review Materials Methods Results Discussion Conclusion and recommendation
  3. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 31. THANK YOU

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