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Molecular Biology Methode in Food Analysis

Molecular Biology Methode in Food Analysis. Prof. Dr. Sudjadi. Genetically Modified Foods. Edited by Prof. Dr. Sudjadi. To what extent does the use of GM foods affect today ’ s society and how will it affect future generations?. GOLDEN RICE.

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Molecular Biology Methode in Food Analysis

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  1. Molecular Biology Methode in Food Analysis Prof. Dr. Sudjadi

  2. Genetically Modified Foods Edited by Prof. Dr. Sudjadi

  3. To what extent does the use of GM foods affect today’s society and how will it affect future generations?

  4. GOLDEN RICE • The addition of 2 genes in the rice genome will complete the biosynthetic pathway • 1. Phytoene synthase (psy) – derived from daffodils • 2. Lycopene cyclase (crt1) – from soil bacteria Erwinia uredovora • Produces enzymes and catalysts for the biosynthesis of carotenoids (β-carotene) in the endosperm Presence of pro-vitamin A gives rice grains a yellowish-orange color, thus, the name ‘Golden Rice’

  5. CODING SEQUENCE PROMOTER poly A signal Plant Selectable Marker Gene Plasmid DNA Construct • bacterial genes • antibiotic marker • replication origin Building the Transgenes ON/OFF Switch Makes Protein stop sign Plant Transgene

  6. Examples of engineered species • Wheat (Triticumaestivum) - a man-made species • Corn (Zea mays) - derived from teosinte • Soybeans (Glycine max) - from G. soja • Potatoes (Solanumtuberosum) - wild varieties are toxic None of the food varieties can grow without help from man

  7. THE MAKING OF A GMO CROP VARIETY Backcrossing and selection (6- 8 generations) x x x Transgenic line Commercial variety Commercial Transgenic Line Biotechnology

  8. Advantages of GM Foods • Pest Resistance • Herbicide Tolerance • Disease Resistance • Drought Tolerance • Cold Tolerance • Nutrition • Pharmaceuticals • Phytomediation • Enhanced taste and quality • Reduced maturation time

  9. Disadvantages of GM Foods • Environmental Hazards • Economical Issues • Human Health Risks • Causes humans to possibly become more allergic to GM goods • Unknown effects on human health

  10. GM crops from foreign countries approved for importing as processed materials in China 1 soybean: GTS 40-3-2 9 corn varieties: Bt-corn Mon810, Bt11,Bt176,Mon 863,NK603,GA21, T25, MON59122,TC1507 7 canola varieties: Ms1Rf1、 Ms1Rf2、 Ms8Rf3、GT73、T45、Oxy235、Tapos 19/ 2

  11. Labelling Policies

  12. GM products should be labeled Three labeling methods: GM raw materials: seeds GM products: containing detectable GM materials GM products: containing non-detectable GM materials

  13. GM-plants and derived foods detection procedure

  14. PCR

  15. LOW HIGH Levels of specificity – GM targets (DNA) plant genomic DNA plant genomic DNA promoter terminator gene Screening targets Gene specific targets Construct specific Event specific targets Arne HA et al., Euro Food Res Tech, 2007

  16. HASIL • Metode berhasil mendeteksi gen ‘ac2’ pada level konsentrasi 0.5% ( tepung Amaranth pada produk kentang) • Pada sampel 0%, yang tidak di spike dengan tepung Amaranth, keberadaan gen ‘ac2’ tidak terdeteksi, yang sesuai dengan ekspektasi peneliti. • Gambar 1.

  17. Gambar 2. menunjukkan tingginya spesifitas primer AcUNI-F/R yang dapat mengamplifikasi produk PCR spesifik yang berkorespondensi dengan gen ‘ac2’, yang hanya ada pada benih Amaranth (kolom1). • Di sisi lain, selain kentang di kolom 2, primer StUNI-F/R mengamplifikasi fragmen PCR dari kedelai ( kolom 7) dan wakil dari family Solanaceae , sweet pepper (kolom8), dan tomat (kolom 9).

  18. dari ketiga produk PCR  disekuence, seperti pd gambar 3. •  konfirmasi kemiripan dengan bagian spesifik gen StTS1 kentang • Pd sweet pepper dan tomat  1 substitusi purin jadi pyrimidine • Kedelai (soybean)  1 delesi, 1 insersi, dan substitusi 22 nukleotida.

  19. 20 produk makanan dr kentang diuji keberadaan dari gen ‘ac2’, amplifikasi dari gen StTS1 dengan ukuran 113 bp terjadi pada seluruh sampel. • Akan tetapi fragmen PCR ukuran 145 bp pada gene’ac2’ tidak teramplifikasi pada seluruh produk ( gambar 4.)

  20. Pada saat yang sama, produk makanan kentang diuji keberadaan promoter CaMV35. •  produk PCR spesifik 105 bp untuk amplifikasi fragmen dari promoter CaMV 35S tidak terjadi pada seluruh sampel, kecuali pada kontrol positif. (gambar 5.)

  21. Dongnong No. 704 Nongyou No. 301 Shennong No. 2 negative control Maohong No. 1 Zhongsu No. 5 Hongza No. 10 Shenfen No. 2 Jiafen No. 1 Jifan No. 4 Xifen No. 3 Shuangfu Zaokuai Lichun Zaofen R144 Tomato seeds Ketchup tomato juice Tomato endogenous reference gene: Lat52 Yashu No. 6 Allelic variation analysis Southern blot analysis confirmed that this gene was single copy in the tested varieties Journal of Agriculture Food Chemistry 2005,50(2):122-125

  22. mixed transgenic non-transgenic no template control mixed seeds A PCaMV35S 195bp Nos Terminator Huafan construct specific fragment B 180bp C CaMV35s promoter Anti-EFE NOS terminator 153bp EcoR I Hind III Screen and construct specific detection of transgenic Huafan No.1 tomato Journal of the Science of Food and Agriculture 85:2159–2166 (2005)

  23. Event-specific PCR Detection of Genetically Modified Soybean GTS 40-3-2 GTS 40-3-2 MON1445 MON531 Non-GM NK603 RT73 GA21 NTC M 1 2 3 4 5 6 7 8 0.05% 0.01% NTC 0.5% 0.1% 5% 3% 1% M 1 2 3 4 5 6 7 8 9 Conc= 10^(-0.323*CT + 12.302) R^2＝0.9993 Specific analysis LOD:2 copies，LOQ: 20 copies。 Standard deviation repeatability and reproducibility were less than 0.2 Sensitivity analysis Coefficient Values of the reference molecule was 0.92 sample analysis

  24. Event-specific PCR detection of two GM-rapeseed （RT73 and T45） A B 0.01% 0.05% NTC 0.5% 0.1% 5% 3% 1% Ms3Rf8 Non-GM Ms1Rf2 RT73 NTC RRS Sensitivity analysis T45 176 B A B LOD andLOQ inevent-specific quantitative PCR detection of event T45 were 1 copy and 10 copiesrespectively;For RT73,LOD and LOQ were 10 copies and 100 copies respectively。 。 Specific analysis Based on the standard curve , rapeseed samples were analysised by quantitative PCR. （A）T45；（B）RT73

  25. Challenges in further GMOs analysis • GMOs labeling threshold and its utilized detection method? • The accuracy of GM contents based on Quantitative RT-PCR? • Validation of CRMs and RMs for approved GMOs in China • Qualitative and quantitative detection for GMOs with stacked- gene? • Communication of GMOs analysis • Develop the GMO detection training course in China

  26. “IDENTITY CARD” for Food for Animal Feed Edited by Prof. Dr. Sudjadi

  27. Horse meat found in salami and pastrami (05/06/2003) The US Government food watchdog is to launch an investigation into salami, chorizo, pastrami and other exotic sausages after an undeclared horse or donkey meat. Food Labelling…… Algerian gang face court over donkey meat scam November 23, 2003 Reuters ALGIERS – “butchers and vets in Algeria have been charged with selling 55 tonnes of donkey meat as beef. “ 'Fraud' over imported food Tip-offs reveal trade in adulterated chicken breastsWednesday December 12, 2001 Chicken breasts contained only 54% chicken, according to a survey conducted by the food standards agency. The rest of their weight consisted of water and hydrolysed Beef and Pork protein added by processors. Friday, 28 March, 2003, Firms warned over food fraud Inspectors discovered beef steaklets containing mechanically recovered chicken in Derbyshire.  Authenticity is a tool for public trust

  28. Thursday, 21 December, 2000, 15:27 GMT MP urges action on meat fraudThe appeal came after the conviction of five people for a multi-million pound meat fraud uncovered by environmental health officers in Rotherham. Panic Over Smuggled Meat Grows27 Nov 2003 NTV-MSNBC Veysi Aslan, a veterinerian said some countries are faced with the illegal importation of buffalo meet and beef. Countries also face the possibility that other meat of unidentified origin is making its way in. ….and traceability Special Reports February 12, 2002 Airport on alert over meat scandalLIVERPOOL Airport was on stand-by amid fears of monkey and ape meat being smuggled into the country. It emerged that 5.5 tonnes of monkey and ape flesh is smuggled into British airports every year.  Traceability is never 100% ensured

  29. Animal Feed Safety UN fears BSE may have spread worldwide 500,000 tons of meal exported Fri, Dec 22, 2000 AP WorldStream Officials kill 1,700 mad elk in effort to stop spread of CWD Monday, December 18, 2000 Alanna Mitchell, research by Ken Rubin Toronto Globe and Mail Sources: WHO / Cervid Council of Canada Japan to ban EU beef, processed beef products Fri, 22 Dec 2000 y Jae Hur Reuters World Report  BSE highlighted the need of Feed species analysis

  30. Identified needs • Guarantee composition and certify authenticity of food and of its raw materials / ingredients • Ensure no cross product contamination (Economic management) • Guarantee compliance with labelling regulations • Increase public trust and brand loyalty

  31. The DNA molecule DNA is present in most biological tissues, whereas proteins and other components may be tissue-specific The DNA molecule is more stable than other molecules The DNA molecules number can be AMPLIFIED with enzymatic means starting from very low quantities

  32. DNA analysis In recent times, the most widely used method for analysing DNA is the Polymerase Chain Reaction, PCR It amplifies the number of DNA molecules by exploiting the mechanism of DNA replication Qualitative PCR allows the identification of a specific sequence, or of more sequences at one time Quantitative PCR allows the estimation of the number of molecules of one specific target, or of more targets at one time Sequencing after PCR allows the determination of the sequence of the amplicons for unambiguous detection

  33. Optimasi Program Suhu Real-Time PCR Kurva Amplifikasi (Fluoresensi vs. Siklus) keterangan Kontrol positif (sampel DNA sapi yang diamplifikasi menggunakan primer yang sudah pasti bisa mengamplifikasi DNA sapi) Sampel DNA sapi yang diamplifikasi menggunakan primer CytbRglu2L dan primer CytbRCb9H Kontrol negatif (reagen supermix tanpa sampel DNA)

  34. Optimasi Program Suhu Real-Time PCR keterangan Sampel DNA sapi yang diamplifikasi menggunakan primer CytbRglu2L dan primer CytbRCb9H Kontrol positif (sampel DNA sapi yang diamplifikasi menggunakan primer yang sudah pasti bisa mengamplifikasi DNA sapi) Kontrol negatif (reagen supermix tanpa sampel DNA)

  35. Application of PCR Lane 1, Chicken; Lane 2, cattle; Lane 3, goat; Lane 4, wild boar; Lane 5, pig; Lane 6-7, (+) control and (-) control cyt b primers used in this work were described by Lenstra et al. (2001) CYT b1 5’-CCATCC AAC ATCTCAGCA TGATGA AA-3’ and CYT b2 5’-GCCCCTCAG AATGATATT TGTCCT CA-3’ Modifyed by Sismindari 29/7/2011 37 International Food Research Journal 18(4): 1489-1491 (2011)

  36. PCR-RFLP kombinasi PCR denganpemotonganensimpadaPCR-product 29/7/2011 Modifyed by Sismindari 38

  37. VALIDATION OF RFLP-COMBINED PCR TECHNIQUE TO DETECT PORCINE CONTAMINATION IN MEATBALL (HALAL ANALYSIS). The 2nd International Conference on Chemical Sciences Proceeding Yogyakarta, October14-16th, 2010 TriJoko Raharjo and Sismindari (1) DNA marker, (2) PorkMB/BamHI, (3) BeefMB/BamHI (4) PorkMB/BseDI, (5) BeefMB/BseDI • DNA marker, (2) Beef meatball (MB), • (3) Pork Meatball 29/7/2011 Modifyed by Sismindari 39

  38. Microorganism Growth in Foods 43

  39. Top five foodborne pathogens • Campylobacter jejuni • The most common cause of foodborne illness. Contracted from raw meats, untreated water. Diarrhea • Salmonella enterica • Contracted from a variety of sources. Diarrhea • Clostridium botulinum • Contracted from home-canned products. Anaerobe. Muscle paralysis/botulism • E.coli O157:H7 • Contracted from a variety of sources. Diarrhea. Renal failure possible. • Listeria monocytogenes • From soil, water, undercooked meats. Listeriosis (non-diarrheal illness)

  40. Detection of Food-Borne Pathogens must be rapid and sensitive methods include: culture techniques – may be too slow immunological techniques - very sensitive molecular techniques probes used to detect specific DNA or RNA sensitive and specific 45

  41. Which methods For the main pathogens of interest, currently available tests are based on (i) microbiological methods with growth and biochemical characterisation (ii) immunological analyses (iii) DNA analyses with PCR, Real-Time PCR, melting analysis, arrays, PNA and several other techniques. Official methods (ISO) only concern microbiological assays. Other essays have been validated but have not yet reached the official standard status. The scientific literature proposes several dozens of approaches and tests, with different degrees of reliability. 46

  42. PROKARYOTE IDENTIFICATION • Various techniques are employed to characterize and identify microorganisms • Phenotypic characteristics • Microscopic morphology • Metabolic differences • Serology • Fatty acid analysis • Genotypic characteristics • Nucleic acid probes • DNA amplification • rRNA sequencing

  43. PHENOTYPIC CHARACTERISTICS Microscopic morphology • Size and shape • Readily determined by microscopic examination of a wet mount • Can determine whether the microbe is a prokaryote, fungus, or protozoan

  44. PHENOTYPIC CHARACTERISTICS Microscopic morphology • Gram stain • Differential stain distinguishing between gram-positive and gram-negative bacteria • Narrows possible identities of an organism • Excludes many possibilities • Generally insufficient alone for diagnosis • e.g., E. coli and Salmonella gram stains look alike

  45. PHENOTYPIC CHARACTERISTICS Metabolic differences • Culture characteristics • MacConkey agar is both selective and differential • Bile salts and dyes inhibit all but certain gram-negative rods • “Selective” • Acid produced by bacteria able to ferment lactose will turn a pH indicator red and form red colonies • “Differential”

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