Thực hiện m-PCR với 6 mẫu bột thịt. Kết quả ở Hình 4.12 cho thấy mẫu 1 có thịt dê, gà, heo, mẫu 2 có thịt dê, gà, mẫu 3 có thịt dê, gà, heo, mẫu 4 có thịt dê, gà, heo, mẫu 5 & 6 không có thịt nào trong 6 loại thịt heo, gà, trâu, bò, dê, cừu.
Hình 4.12: Kết quả m-PCR với bột thịt trên thị trường
1 2 3 Lad 4 5 6 ( -)
500bp
Chương 5
KẾT LUẬN VÀ ĐỀ NGHỊ 5.1 Kết luận
1. Đã hoàn thiện quy trình m-PCR phát hiện thịt heo, gà, dê, cừu, bò lẫn trâu với tỷ lệ thích hợp của các primer FSIM:RG:RCh:RCB:RS:RP là 12: 7: 7:10: 5: 25 pmol. Quy trình này có thể áp dụng cho thịt tươi, và thịt xử lý nhiệt ở 80oC/15’; 120oC/15’; 120oC/30’; 130oC/15’; 130oC/30’, 180oC/15’. Ảnh hưởng của tỷ lệ DNA hiện diện trong các hỗn hợp DNA và hỗn hợp thịt lên khả năng phát hiện của m-PCR cũng được xác định.
2. Phân biệt loài của thịt trong sản phẩm thịt chế biến bằng phương pháp multiplex - PCR bị ảnh hưởng bởi thời gian, nhiệt độ, và kích thước của đoạn DNA mục tiêu.
5.2 Đề nghị
1. Tiến hành thí nghiệm m-PCR phát hiện các loài trâu/& bò, dê, cừu với tỷ lệ cao hơn 10% khi được xử lý ở 130oC/30’ để xác định ở 130oC/30’ thì tỷ lệ thấp nhất của trâu lẫn bò, dê, cừu mà m-PCR phát hiện được.
2. Ứng dụng m-PCR để khảo sát thêm nhiều hỗn hợp thịt chế biến và nhiều mẫu bột thịt hơn nữa.
TÀI LIỆU THAM KHẢO TIẾNG VIỆT
1. Bùi Thị Cúc, 2006. Tình hình xuất nhập khẩu động vật và các sản phẩm động vật. Cục Thú y
2. Hồ Huỳnh Thùy Dương, 2002. Sinh học phân tử. Nhà xuất bản Giáo dục Thành
phố Hồ Chí Minh
3. Hồ Thị Nguyệt Thu, 2003. Chế biến thịt. Giáo trình học tập trường Đại học Nông Lâm TP. HCM
4. Lương Quý Phương, 2006. Sản xuất bộ kit tách chiết DNA và bộ kit PCR phát hiện gen halothan trên heo. Khóa luận tốt nghiệp kỹ sư công nghệ sinh học. Trường Đại học Nông Lâm TP HCM.
5. Lưu Phúc Lợi, 2005. Bài giảng Sinh tin học. Đại học Nông Lâm TPHCM
6. Nguyễn Ngọc Tuân, Lê Thanh Hiền, 2004. Chế biến bảo quản thịt và sữa. Nhà xuất bản Nông Nghiệp Tp. Hồ Chí Minh, 163 trang
7. Trịnh Thị Thanh Huyền, 2007. Phân biệt các loại thịt heo, bò, cừu bằng phương pháp multiplex PCR. Khóa luận tốt nghiệp kỹ sư công nghệ sinh học. Trường Đại học Nông Lâm TP HCM.
TIẾNG NƯỚC NGOÀI
8. Alberts B., Bray D., Lewis J., Raf M., Roberts K. and Watson J.D.,1994.
Molecular Biology of the Cell (3rd ed.), Garland Publishing Inc, New York and London
9. Arslan A., Ilhak O. I. Calicioglu M., 2006. Effect of method of cooking on dentification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Science 72: 326–330
10. Ashoor S. H., Monte W. G., Stiles P. G., 1998. Liquid chromatographic identification of meats. Journal of Association of Official Analytical Chemists 71:397–403.
11. Behrens M., Untham., Brinkmann Y., Buchholz R., and Latus N.,1999. Identification of Animal Species in Heated and Complex Meat Products Using Species Specific PCR Reactions. Fleischwirtsch 79: 97 – 100.
12. Bellagamba F., Valfre F., Panseri S., & Moretti V. M., 2003. Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal.
Journal of Food Protection 66(4): 682–685.
13. Beneke B. and Hagen M., 1998. Applicability of PCR (Polymerase Chain Reaction) for the Detection of Animal Species in Heated Meat Products.
Fleischwirts 78: 1016 – 1019.
14. Bogenhagen D., Clayton D.A., 1974. The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Journal of Biological Chemistry 249:7791.
15. Borgo R., Souty Grosset C., Bouchon & Gomot D. L., 1996. PCR-RFLP analysis of mitochondrial DNA for identification of quail meat species. Journal of Food Science 61: 1–4.
16. Bossier P., 1999. Authentication of seafood products by DNA pattern. Journal Food Science 64:1899–1993.
17. Brodmann P. D., Moor D., 2003. Sensitive and semi quantitative TaqMan real time polymerase chain reaction systems for the detection of beef (Bos taurus) and the detection of the family mammalia in food and feed. Meat Science 65:599–607.
18. Brodmann P. D., Nicholas G., Schaltenbrand P., Ilg E. C., 2001. Identifying unknown game species: experience with nucleotide sequencing of the mitochondrial cytochrome b gene and a subsequent basic local alignment search tool search. European Food Research and Technology 212:491– 496.
19. Burgener M., Hubner P.,1998. Mitochondrial DNA enrichment for species identification and revolutionary analysis. Z Lebensm Unters Forsch
207:261–263.
20. Carnegie P. R., Illic M. Z., Etheridge M.O., Collins M. G., 1983. Improved high performance liquid chromatographic method for analysis of histidine dipeptides anserine, camosine and balenine present in fresh meat. Journal of Chromatography 261:153–157.
21. Chikuni K., Tabata T., Kosugiyama M., Monma M. and Saito M., 1994. Polymerase Chain Reaction Assay for Detection of Sheep and Goat Meats.
22. Chow S., Inogue S., 1993. Intra- and Interspecifc Restriction Fragment Length Polymorphism in Mitochondrial Genes of Thun- nus Tuna Species.
National Research Institute of FarSeasFisheries 30: 207-224.
23. Chung G.S., Lee M. H., Kim J. M., Park J. M., 1998. Differentiation the species of origin of meats on the basis of the contents of histidine dipeptides in muscle. Journal of Veterinary Science 40:1–6.
24. Collins S.J., Lawson V. A. Masters C.L., 2004. Transmissible spongiform encephalopathies. Lancet 363 (9402): 51–61.
25. Colombo F., Viacava R., Giaretti M., 2000. Differentiation of the species ostrich (Struthio camelus) and emu (Dromaius novaehollandiae) by polymerase chain reaction using an ostrich-specific primer pair. Meat Science 56:15–17. 26. Cushwa W. T., Medrano J. F., 1996. Applications of the random amplified polymorphic DNA (RAPD) assay for genetic analysis of livestock species.
Animal Biotechnology 7:11–31.
27. Dalmasso A., Fontanella E., Piatti P., Civera T., Rosati S., Bottero M.T., 2004. A multiplex PCR assay for the identification of animal species in feedstuffs.
Molecular and Cellular Probes 18: 81–87.
28. Desmarais E., Lanneluc I., Lagne J., 1998. Direct amplification of length polymorphism (DALP) or how to get and characterize new genetic markers in many species. Nucleic Acids Research 26:1458–1465.
29. Ellen H., Hans T., Gottfried S., 1964. Deoxyribonucleic Acid Associated with Yeast Mitochondria. Biochemical and Biophysical Research Communication 15: 127 - 132.
30. Fairbrother K. S., Hopwood A. J., Lockley A. K., Bardsley R. G., 1998. Meat speciation by restriction fragment length polymorphism analysis using an - actin cDNA probe. Meat Science 50:105–114.
31. Fairbrother K.S., Hopwood A.J., Lockley A.K. and Bardsley R.G., 1998. The Actin Multigene Family and Livestock Speciation Using the Polymerase Chain Reaction. Animal Biotechnology 9: 89 – 100.
32. Fontaine K.M., Cooley J.R., Simon C., 2007. Evidence for paternal leakage in hybrid periodical cicadas (Hemiptera: Magicicada spp.). PloS one 2(9): e892.
33. Fukall L., Kas J., 1989. The advantages of immunoassay in food analysis. Trends in Analytical Chemistry 8: 112 – 116.
34. Guoli Z., Mingguang Z., Zhijiang Z., Hongsheng O., Qiang L., 1999. Establishment and application of a polymerase chain reaction for the identification of beef. Meat Science 51:233–236.
35. Gyllesten, U. B., Wharton, D., Josefsson, A. and Wilson, A. C., 1991. Paternal inheritance of mitochodrial DNA in mice. Nature 352: 255-257.
36. Haushi S., Basumatary R., Girish P. S., Doley S., Bardoloi R. K., Kumar A., 2009. Identification of chicken, duck, pigeon and pig meat by species- specific markers of mitochondrial origin. Meat science. Article in Press. 37. Hayashi K., 1996. PCR SSCP. Single Strand Conformation Polymorphism
Analysis of PCR Products. Laboratory Proto- cols for Mutation Detection (Landegren, U., ed.), Oxford University Press. pp. 14 – 22.
38. Herman L., 2000. Determination of the animal origin of raw food by species- specific PCR. Journal of Dairy Research 68:429–436.
39. Hird H., J., Chisholm A.,Sanchez M., Hernandez R., Goodier K., Schneede C., Boltz and Popping B., 2006. Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real- time polymerase chain reaction. Food Additives and Contaminants. 23:645–650
40. Hitchcock C. H. S., 1998. Immunoassays for veterinary and food analysis.
Elsevier Applied Science Publishers Ltd., London, New York, p3
41. Hoeh W.R., Blakley K.H., Brown W.M., 1991. Heteroplasmy suggests limited biparental inheritance of Mytilus mitochondrial DNA. Science 251:1488– 1490.
42. Hofmann K., 1997. Nachweis der Tierart bei Fleisch und Fleischerzeugnissen. 2. Mitteilung. Fleischwirtschaft 77 (2):151-154. In: Detection of meat of different animal species in meat products. P. 2. Review of applicable methods. Mieso i Wedliny 1998, 3, 74-79 [in Polish]
43. Holland P. M., Abramson R. D., Watson R., Gelfand D. H., 1991. Detection of specific polymerase chain reaction product by utilization the 5_ to 3_ exonuclease activity of Thermus aquaticus. Proceedings of the National
Academy of Sciences USA 88:7276–7280.
44. Hong W., Gao D. B., Zhang A. H., Pan W. Q., Lian, C. Z. L., 2004. Multiplex Polymerase Chain Reaction Method for Detection of Bovine Materials in Foodstuffs. Journal of Association of Official Analytical Chemists International. 86(4): 764-767.
45. Hopwood A.J., Fairbrother K.S., Lockley A.K. and Bardsley R.G., 1999. An Actin Gene-Related Polymerase Chain Reaction (PCR) Test for Identification of Chicken in Meat Mixtures. Meat Science. 53: 227 – 231. 46. Hsieh H.M., Chiang H.L., Tsai L.C., Lai S.Y., Huang N.E., Linacre A., Lee
J.C.I., 2001. Cytochrome b gene for species identification of the conservation animals. Forensic Science International 122: 7-18.
47. Hsing M. H., Chin C. T., Li C. T., Hsiao L. C., Nu E. H., Rocky T. P. S., Adrian L., James C. L., 2005. Species identification of meat products using the cytochrome b gene. Forensic science Journal 4: 29-36.
48. Hui Y. H., 2006. Handbook of Food science technology and engineering. CRC Press Taylor & Francis Group.
49. Irfan O. I., 2007. Identification of meat species by polymerase chain reaction (PCR) teachnique. Turkish Journal of Veterinary and Animal Sciences 31(3): 159-163.
50. Irwin D.M., Kocher T.D., Wilson A.C., 1991. Evolution of the cytochrome b gene of mammals. Journal of Molecular Evolution 32: 128-44
51. Jain S., Brahmbhait M. N., Rank D. N., Joshi C. G.. and Solank J. V., 2007. Use of cytochrome b gene variability in detecting meat species by multiplex PCR assay. Indian Journal of Animal Sciences 77 (9): 880-881.
52. Kesmen Z., Sahin F., Yetim H., 2007. PCR assay for the identification of animal species in cooked sausages. Meat Science 77: 649–653
53. Kocher T. D., Thomas W. K., Meyer A., Edwards S. V., Paabo S., Villablanca F. X., et al., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing of conserved primers. Proceedings of the National Academy Sciences of the United States of America 86: 6196-200. 54. Koh M. C., Lim C. H., Chua S. B., Chew S. T., Phang S. T. W., 1998. Random
meat animal species. Meat Science 48:275–285.
55. Kondo R., Matsuura E.T., Chigusa S.I., 1992. Further observation of paternal transmission of Drosophila mitochondrial DNA by PCR selective amplification method. Genetical Research 59 (2): 81–4)
56. Kremar P. and Rencova E., 2003. Identification of species specific DNA in feedstuffs. Journal of Agricultural and Food Chemistry. 51(26): 7655- 7658.
57. Lahiff S., Glennon M., Lyng J., Smith T, Shilton N., Maher M., 2002. Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples. Journal of Food Protection 65:1158–1165.
58. Laube L., Spiegelberg A., Butschke A., Zagon J., Schauzu M., Kroh L., Broll H., 2003. Methods for the detection of beef and pork in foods using real-time polymerase chain reaction. International Journal of Food Science &
Technology 38:111–118.
59. Lee J.C.I. and Chang J.G., 1994. Random Amplified Poly-morphic DNA Polymerase Chain Reaction (RAPD PCR) Finger-prints in Forensic Species Identification. Forensic Science International 67: 103 – 107.
60. Lockley A. K., Bardsley R. G., 2000. DNA-based methods for food authentication. Trends in Food Science & Technology. 11: 67 – 77.
61. Maede, D., 2006. A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences. European Food Research and Technology 224(2): 209–217.
62. Martin, I., Garcia T., Fajardo V., Lopez-Calleja I., Hernández P. E., Gonzalez I., and Martín R.. 2007. Species-specific PCR for the identification of ruminant species in feedstuffs. Meat Science. 75:120–127.
63. Martinez I., 1997. DNA typing of fish products for species identification. In: Seafood from Product to Consumer: Integrated Approach to Quality. J Luten, T Borresen, J Oehlenschlager (eds.). Amsterdam: Elsevier Science
497–506.
64. Martinez I., Yman M., 1999. Species identification in meat products by RAPD analysis. Food Research International 31:459–466.
65. Matsunaga T., Chikuni K., Tanabe R., Muroya S., Shibata K., Yamad J., Shinmura Y., 1999. A quick and simple method for the identification of meat species cow meat product by PCR assay. Meat Science 51: 143-148. 66. McPherson M. J. and Moller S. G., 2006. PCR. Taylor and Francis Group.
67. Meusel M.S., Moritz R.F., 1993. Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs. Cureent Genetics. 24 (6): 539–43).
68. Meyer R., Candrian U. and Luethy J., 1994. Detection of Pork in Heated Meat Products by Polymerase Chain Reaction (PCR). Journal AOAC International 77: 617 – 622.
69. Meyer R., Hoefelein C., Luethy J., & Candrian U., 1995. Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food. Journal of Association of Official Analytical Chemists International 78: 1542–1551.
70. Michael A. I., David H. G., 1990. PCR protocol: A guide to methods and application. Academic Press, San Diego, CA. p 3-12.
71. Michele L., 2003. Food authenticity traceability. Woodhead Publishing Limited Abington Hall, Abington Cambridge CB1 6AH. England.
72. Miguel A. R., Teresa G., Isabel G., Luis A., Pablo E. H. and Rosario M., 2004. PCR identification of beef, sheep, goat and pork in raw and heat treated meat mixtures. Journal of Food Protection 67: 172-77.
73. Minkiewicz P., Dziuba J., Nałecz D., 2000. Modern methods of separation and research of peptides structure and proteins of food. Przem. Spo 12: 34-37. 74. Montiel-Sosa J.F., Ruiz E., Montoya J., Roncales P., Lopez-Perez M.J., Perez-
Martos A., 2000. Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA.
Journal of Agricultural and Food Chemistry 48(7):2829-2832.
75. Murray B. W., McClymont R. A., & Strobeck C., 1995. Forensic identification of ungulate species using restriction fragment digests of PCR amplified mitochondrial DNA. Journal of Forensic Science 40: 943–951.
76. Nass M.M., Nass S., 1963. Intramitochondrial Fibers with DNA characteristics.
77. Partis L., Croan D., Guo Z., Clark R., Coldham T. and Murby J.,2000. Evaluation of a DNA Fingerprinting Method for Determining the Species of Origin of Meats. Meat Science 54: 369 – 376.
78. Pascoal A., Prado M., Calo P., Cepeda A., & Barros-Velazquez J., 2005. Detection of bovine DNA in raw and heat-processed foodstuffs, commercial foods and specific risk materials by a novel specific polymerase chain reaction method. European Food Research and Technology 220(3–4): 444–450
79. Penman & Danny, 2002. Mitochondria can be inherited from both parents.
NewScientist.com. 5/2/2008.
80. Pinto A. D., Forte V.T., Conversano M.C., Tantillo G.M., 2005. Duplex polymerase chain reaction for detection of pork meat in horse meat fresh sausages from Italian retail sources. Food Control 16: 391-394.
81. Pyz L. R., 1998. Methods of species identification of meat. Med. Wet. 54 (11): 732-736 [in Polish].
82. Rahman A. S. M., Ahmed M. M. M., 2007. Rapid and sensitive identification of buffalo’s, cattle’s and sheep’s milk using species-specific PCR and PCR– RFLP techniques. Food Control 18:1246–1249
83. Rea S., Chikuni K., Avellini P.,1996. Possibility of using single strand conformation polymorphism (SSCP) analysis for discriminating European pig and wild boar meat samples. Italian Journal of Food Science 3:211– 220.
84. Roa K.B.C.A., Bhat V. and Totey S.M., 1996. Detection of Species-Specific Genetic Markers in Farm Animals Through Random Amplified Polymorphic DNA (RAPD). Genetic Analysis: Biomolecular Engineering
13: 135 – 138.
85. Rodriguez M. A., Garcia T., Gonzalez I., Asensio L., Mayoral B., Lopez-Calleja I., Hernandez P. E., Martin R., 2003. Identification of goose, mule duck, turkey, and swine in foie grass by species-specific polymerase chain reaction. Journal of agriculture and food chemistry 51:1524–1529.
86. Saeed T., Ali S. G., Rahman H. A.A., Sawaya W. N., 1989. Detection of pork and lard as adulterants in processed meat: Liquid chromatographic analysis of derivatized triglycerides. Journal - Association of Official Analytical
Chemists 72:921–925, 1989.
87. Saez R., Sanz Y., Toldra F., 2004. PCR-based fingerprinting techniques for rapid detection of animal species in meat products. Meat Science 66:659–665. 88. Sambrook J., Russell W. D., 2001. Molecular cloning (A laboratory manual).
Cold Spring Harbor laboratory press, New York, USA.
89. Sawyer M., Rensen G., Smith W., Yee M., Wong A., Osburn B., Cullor J., 1997.
Overcoming RNA inhibition in the fluorescent polymerse chain reaction assay to enhance detection of bovine DNA in cattle feeds. Department of Population Health and Reproduction, School of Veterinary Medicine, University of California.
90. Sutovsky, P., et. al, 1999. Ubiquitin tag for sperm mitochondria. Nature 402: 371–372
91. Tajima K., Enishi O., Amari M., Mitsumori M., Kajikawa H., Kurihara M., Yanai S., Matsui H., Yasue H., Mitsuhashi T., Kawashima T., Matsumoto M., 2002. PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements.
Bioscience, Biotechnology, and Biochemistry 66(10):2247–2250.
92. Tartaglia M., Saulle E., Pestalozza S., Morelli L., Antonucci G., Battaglia P., 1998. Detection of bovine mitochondrial DNA in ruminant feeds: A