molecular biology methode in food analysis prof. dr. sudjadi
TRANSCRIPT
Molecular Biology Methode Molecular Biology Methode in Food Analysisin Food Analysis
Prof. Dr. SudjadiProf. Dr. Sudjadi
Genetically Modified FoodsGenetically Modified Foods
Edited by Prof. Dr. SudjadiEdited by Prof. Dr. Sudjadi
To what extent does the use of GM To what extent does the use of GM foods affect today’s society and how foods affect today’s society and how
will it affect future generations?will it affect future generations?
GOLDEN RICEGOLDEN RICE
The addition of 2 genes in the rice The addition of 2 genes in the rice genome will complete the genome will complete the biosynthetic pathwaybiosynthetic pathway– 1. Phytoene synthase (psy) – 1. Phytoene synthase (psy) –
derived from daffodilsderived from daffodils– 2. Lycopene cyclase (crt1) – from 2. Lycopene cyclase (crt1) – from
soil bacteria soil bacteria Erwinia uredovoraErwinia uredovora
Produces enzymes and catalysts Produces enzymes and catalysts for the biosynthesis of carotenoids for the biosynthesis of carotenoids ((ββ-carotene) in the endosperm-carotene) in the endosperm
Presence of pro-vitamin A gives rice grains a yellowish-Presence of pro-vitamin A gives rice grains a yellowish-orange orange colorcolor, thus, the name , thus, the name ‘‘Golden RiceGolden Rice’’
CODING SEQUENCE poly A signalPROMOTER
Building the Transgenes
Plant Transgene
bacterial genes•antibiotic marker•replication origin
Plant Selectable Marker Gene
Plasmid DNA Construct
ON/OFF Switch Makes Protein stop sign
Examples of engineered speciesExamples of engineered species
Wheat (Wheat (Triticum aestivumTriticum aestivum) - a man-made ) - a man-made speciesspecies
Corn (Corn (Zea maysZea mays) - derived from teosinte) - derived from teosinte
Soybeans (Soybeans (Glycine maxGlycine max) - from ) - from G. sojaG. soja
Potatoes (Potatoes (Solanum tuberosumSolanum tuberosum) - wild ) - wild varieties are toxicvarieties are toxic
None of the food varieties can grow without help from man
THE MAKING OF A GMO CROP VARIETY
Backcrossing and selection (6- 8 generations)
Transgenic line
Commercial variety
x
x
x
Commercial Transgenic LineBiotechnology
Advantages of GM FoodsAdvantages of GM Foods
1.1. Pest ResistancePest Resistance2.2. Herbicide ToleranceHerbicide Tolerance3.3. Disease ResistanceDisease Resistance4.4. Drought ToleranceDrought Tolerance5.5. Cold ToleranceCold Tolerance6.6. NutritionNutrition7.7. PharmaceuticalsPharmaceuticals8.8. PhytomediationPhytomediation9.9. Enhanced taste and qualityEnhanced taste and quality10.10.Reduced maturation timeReduced maturation time
Disadvantages of GM FoodsDisadvantages of GM Foods
1.1. Environmental Hazards Environmental Hazards
2.2. Economical IssuesEconomical Issues
3.3. Human Health RisksHuman Health Risks– Causes humans to possibly become more Causes humans to possibly become more
allergic to GM goodsallergic to GM goods– Unknown effects on human healthUnknown effects on human health
GM crops from foreign countries approved for GM crops from foreign countries approved for
importing as processed materials in Chinaimporting as processed materials in China
1 soybean:1 soybean: GTS 40-3-2 GTS 40-3-2
9 corn varieties:9 corn varieties: Bt-corn Mon810, Bt11,Bt176,Mon Bt-corn Mon810, Bt11,Bt176,Mon
863,NK603,863,NK603, GA21, T25, MON59122,GA21, T25, MON59122, TC1507TC1507
7 canola varieties:7 canola varieties: Ms1Rf1 Ms1Rf1 、 、 Ms1Rf2Ms1Rf2 、 、 Ms8Rf3Ms8Rf3 、、 GT73GT73 、、 T45T45 、、 Oxy235Oxy235 、、 Tapos 19/ 2 Tapos 19/ 2
Countries/Regions Type of Policy GM Content Threshold
Australia/New Zealand Mandatory 1%
China Mandatory n/a
Czech Mandatory 1%
European Union/UK Mandatory 0.9%
Hong Kong Mandatory 5%
Japan Mandatory 5%
Russia Mandatory 5%
Switzerland Mandatory2% or 3% depending on
different situations
Brazil (draft) Mandatory n/a
India (draft) Mandatory n/a
Israel (draft) Mandatory 1%
Malaysia (draft) Mandatory 3%
Korea (draft) Mandatory 3%
Taiwan (draft) Mandatory 5%
Thailand (draft) Mandatory 5%Canada Voluntary 5%USA Voluntary 5%
* Sources: International Service for the Acquisition of Agri-biotech Applications
(http://www.isaaa.org) and the United State Department of Agriculture
(http://www.fas.usda.gov)
Labelling Policies
GM products should be labeledGM products should be labeled
SoybeanSoybean seed, soybean, flour, oil, soy mealseed, soybean, flour, oil, soy meal
CornCorn seed, corn, flour, oil, corn mealseed, corn, flour, oil, corn meal
RapeseedRapeseed seed, rapeseed, oil, rapeseed mealseed, rapeseed, oil, rapeseed meal
TomatoTomato seed, fresh tomato, tomato sauceseed, fresh tomato, tomato sauce
Three labeling methods:
GM raw materials: seeds
GM products: containing detectable GM materials
GM products: containing non-detectable GM materials
GM-plants and derived foods detection GM-plants and derived foods detection procedureprocedure
Samples
Tested Samples Saved Samples
Protein Detection Methods
Nucleic Acids Detection Methods
ELISA Lateral Flow StripDNA Extraction
Conventional PCR
Contained GM contents
No GM contents
Quantitative PCR
GM Contents (xx%)
Sampling
Negative Positive
Samples
Tested Samples Saved Samples
Protein Detection Methods
Nucleic Acids Detection Methods
ELISA Lateral Flow StripDNA Extraction
Conventional PCR
Contained GM contents
No GM contents
Quantitative PCR
GM Contents (xx%)
Sampling
Negative Positive
plant genomic DNA plant genomic DNApromoter gene terminator
Screening targets
Gene specific targets
Construct specific
Event specific targets
Levels of specificity – GM targets (DNA)
LOW
HIGHArne HA et al., Euro Food Res Tech, 2007
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.
• 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).
• 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.
• 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.)
• 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.)
Tomato seeds Ketchup tomato juice
Journal of Agriculture Food Chemistry 2005,50(2):122-125
Southern blot analysis confirmed that this gene was single copy in the tested varieties
Allelic variation analysis
Tomato endogenous reference gene: Lat52
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No. 3
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ong
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Screen and construct specific detection of transgenic Huafan No.1 tomato
CaMV35s promoter
Anti-EFE
NOS terminator
EcoR I Hind III
Journal of the Science of Food and Agriculture 85:2159–2166 (2005)
PCaMV35S
Nos Terminator
Huafan construct specific fragment
A
B
C
195bp
180bp
153bp
mix
ed tr
ansg
enic
mix
ed s
eeds
non-
tran
sgen
ic
no te
mpl
ate
cont
rol
M 1 2 3 4 5 6 7 8
Specific analysisSpecific analysis
M 1 2 3 4 5 6 7 8 9
Event-specific PCR Detection of Genetically Modified Event-specific PCR Detection of Genetically Modified Soybean Soybean GTS 40-3-2GTS 40-3-2
NTC
NTCNon
-GM
Non
-GM
MO
N53
1
MO
N53
1
GA
21G
A21
MO
N14
45
MO
N14
45R
T73
RT7
3
NK
603
NK
603
GTS
40-
3-2
GTS
40-
3-2
5%5% 3%3% 1%1% 0.5%
0.5%
0.1%
0.1% 0.05
%0.
05%
0.01
%0.
01%
NTC
NTC
Conc= 10^(-0.323*CT + 12.302)R^2 = 0.9993
Sensitivity analysisSensitivity analysis
LOD:2 copiesLOD:2 copies ,, LOQ: 20 copiesLOQ: 20 copies 。。Standard deviation repeatability andStandard deviation repeatability and
reproducibility were less than reproducibility were less than 0.20.2Coefficient Values of the Coefficient Values of the reference moleculereference molecule was was 0.92 0.92 sample sample
analysisanalysis
Event-specific PCR detection of two GM-rapeseed Event-specific PCR detection of two GM-rapeseed (( RT73 and T45RT73 and T45 ))
Specific analysisSpecific analysis
(( AA )) T45T45 ;(;( BB )) RT73RT73
LOD andLOD and LOQ LOQ inin event-specific quantitative event-specific quantitative
PCR detection of event PCR detection of event T45 T45 were were 11 copy copy andand
10 10 copiescopies respectively;respectively;For For RT73,RT73, LOD and LOD and
LOQ LOQ werewere 10 10 copiescopies and 100 and 100 copies copies
respectivelyrespectively 。。
。。
NTC
NTC Non
-GM
Non
-GM
T45
T45 Ms3
Rf8
Ms3
Rf8
176
176
RT7
3R
T73
Ms1
Rf2
Ms1
Rf2
RR
SR
RS
AA
BB
Sensitivity analysisSensitivity analysis
5%5% 3%3% 1%1% 0.5%
0.5%
0.1%
0.1%
0.05
%0.
05%
0.01
%0.
01%
NTC
NTC
AA
BB
BB
Based on the standard curve , rapeseed Based on the standard curve , rapeseed samples were analysised by quantitative samples were analysised by quantitative PCR.PCR.
Challenges in further GMOs analysisChallenges 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
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 breasts Wednesday 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.
Food Labelling……
Friday, 28 March, 2003, Firms warned over food fraudInspectors discovered beef steaklets containing mechanically recovered
chicken in Derbyshire.
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.
Authenticity is a tool for public trustAuthenticity is a tool for public trust
Thursday, 21 December, 2000, 15:27 GMT MP urges action on meat fraud
The appeal came after the conviction of five people for a multi-million pound meat fraud uncovered by environmental health
officers in Rotherham.
….and traceability
Special Reports February 12, 2002 Airport on alert over meat
scandal LIVERPOOL 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.
Panic Over Smuggled Meat Grows
27 Nov 2003 NTV-MSNBCVeysi 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.
Traceability is never 100% ensured Traceability is never 100% ensured
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 CWDMonday, December 18, 2000 Alanna Mitchell, research by Ken
Rubin Toronto Globe and MailSources: WHO / Cervid Council of Canada
Japan to ban EU beef, processed beef productsFri, 22 Dec 2000 y Jae Hur Reuters World Report
Animal Feed Safety
BSE highlighted the need of Feed species analysisBSE highlighted the need of Feed species analysis
Guarantee composition and certify authenticityGuarantee composition and certify authenticityof food and of its raw materials / ingredientsof food and of its raw materials / ingredients
Ensure no cross product contamination (Economic Ensure no cross product contamination (Economic management)management)
Guarantee compliance with labelling regulationsGuarantee compliance with labelling regulations
Increase public trust and brand loyaltyIncrease public trust and brand loyalty
Identified needs
The DNA moleculeThe DNA molecule
DNA is present in most biological DNA is present in most biological tissues, whereas proteins and other tissues, whereas proteins and other components may be tissue-specificcomponents may be tissue-specific
The DNA molecule is more stable The DNA molecule is more stable than other moleculesthan other molecules
The DNA molecules number can be The DNA molecules number can be AMPLIFIED with enzymatic means AMPLIFIED with enzymatic means starting from very low quantitiesstarting from very low quantities
DNA analysisDNA analysis
In recent times, the most widely used method for In recent times, the most widely used method for analysing DNA is the Polymerase Chain Reaction, analysing DNA is the Polymerase Chain Reaction, PCR PCR
It amplifies the number of DNA molecules by It amplifies the number of DNA molecules by exploiting the mechanism of DNA replicationexploiting the mechanism of DNA replication– Qualitative PCR allows the identification of a specific Qualitative PCR allows the identification of a specific
sequence, or of more sequences at one timesequence, or of more sequences at one time– Quantitative PCR allows the estimation of the number of Quantitative PCR allows the estimation of the number of
molecules of one specific target, or of more targets at one molecules of one specific target, or of more targets at one timetime
– Sequencing after PCR allows the determination of the Sequencing after PCR allows the determination of the sequence of the amplicons for unambiguous detectionsequence of the amplicons for unambiguous detection
Kurva Amplifikasi (Fluoresensi vs. Siklus)
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 CytbRCb9HKontrol negatif (reagen supermix tanpa sampel DNA)
keterangan
Sampel DNA sapi yang diamplifikasi menggunakan primer CytbRglu2L dan primer CytbRCb9HKontrol positif (sampel DNA sapi yang diamplifikasi menggunakan primer yang sudah pasti bisa mengamplifikasi DNA sapi)Kontrol negatif (reagen supermix tanpa sampel DNA)
keterangan
29/7/2011Modifyed by Sismindari 37
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’
Lane 1, Chicken; Lane 2, cattle; Lane 3, goat; Lane 4, wild boar; Lane 5, pig; Lane 6-7, (+) control and (-) control
International Food Research Journal 18(4): 1489-1491 (2011)
Application of PCR
29/7/2011 Modifyed by Sismindari 39
The 2nd International Conference on Chemical Sciences Proceeding
Yogyakarta, October14-16th, 2010
TriJoko Raharjo and Sismindari
VALIDATION OF RFLP-COMBINED PCR TECHNIQUE TO DETECT PORCINE CONTAMINATION IN MEATBALL (HALAL ANALYSIS).
(1) DNA marker, (2) Beef meatball (MB), (2) (3) Pork Meatball
(1) DNA marker,
(2) PorkMB/BamHI,
(3) BeefMB/BamHI
(4) PorkMB/BseDI,
(5) BeefMB/BseDI
Top five foodborne pathogensTop five foodborne pathogensCampylobacter jejuniCampylobacter jejuni– The most common cause of foodborne illness. Contracted from raw The most common cause of foodborne illness. Contracted from raw
meats, untreated water. Diarrheameats, untreated water. Diarrhea
Salmonella entericaSalmonella enterica– Contracted from a variety of sources. DiarrheaContracted from a variety of sources. Diarrhea
Clostridium botulinumClostridium botulinum– Contracted from home-canned products. Anaerobe. Muscle Contracted from home-canned products. Anaerobe. Muscle
paralysis/botulismparalysis/botulism
E.coli E.coli O157:H7O157:H7– Contracted from a variety of sources. Diarrhea. Renal failure possibleContracted from a variety of sources. Diarrhea. Renal failure possible ..
Listeria monocytogenesListeria monocytogenes– From soil, water, undercooked meats. Listeriosis (non-diarrheal From soil, water, undercooked meats. Listeriosis (non-diarrheal
illness)illness)
4545
Detection of Food-Borne PathogensDetection of Food-Borne Pathogens
must be rapid and sensitivemust be rapid and sensitive
methods include:methods include:– culture techniques – may be too slowculture techniques – may be too slow– immunological techniques - very sensitiveimmunological techniques - very sensitive– molecular techniquesmolecular techniques
probesprobes used to detect specific DNA or RNA used to detect specific DNA or RNA
sensitive and specificsensitive and specific
4646
Which methodsWhich methods
For the main pathogens of interest, currently available tests For the main pathogens of interest, currently available tests are based onare based on– (i) microbiological methods with growth and biochemical (i) microbiological methods with growth and biochemical
characterisationcharacterisation– (ii) immunological analyses(ii) immunological analyses– (iii) DNA analyses with PCR, Real-Time PCR, melting (iii) DNA analyses with PCR, Real-Time PCR, melting
analysis, arrays, PNA and several other techniques. analysis, arrays, PNA and several other techniques. Official methods (ISO) only concern microbiological assays. Official methods (ISO) only concern microbiological assays. Other essays have been validated but have not yet reached Other essays have been validated but have not yet reached the official standard status. the official standard status. The scientific literature proposes several dozens of The scientific literature proposes several dozens of approaches and tests, with different degrees of reliability. approaches and tests, with different degrees of reliability.
PROKARYOTE IDENTIFICATIONPROKARYOTE IDENTIFICATION
Various techniques are employed to Various techniques are employed to characterize and identify microorganismscharacterize and identify microorganisms– Phenotypic characteristicsPhenotypic characteristics
Microscopic morphologyMicroscopic morphologyMetabolic differencesMetabolic differencesSerologySerologyFatty acid analysisFatty acid analysis
– Genotypic characteristicsGenotypic characteristicsNucleic acid probesNucleic acid probesDNA amplificationDNA amplificationrRNA sequencingrRNA sequencing
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
Microscopic morphologyMicroscopic morphology
Size and shapeSize and shape– Readily determined by microscopic Readily determined by microscopic
examination of a wet mountexamination of a wet mount– Can determine whether the microbe is a Can determine whether the microbe is a
prokaryote, fungus, or protozoanprokaryote, fungus, or protozoan
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
Microscopic morphologyMicroscopic morphology
Gram stainGram stain– Differential stain Differential stain
distinguishing between distinguishing between gram-positive and gram-positive and gram-negative bacteriagram-negative bacteria
– Narrows possible identities of an organismNarrows possible identities of an organismExcludes many possibilitiesExcludes many possibilities
– Generally insufficient alone for diagnosisGenerally insufficient alone for diagnosise.g., e.g., E. coliE. coli and and SalmonellaSalmonella gram stains look alike gram stains look alike
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
Metabolic differencesMetabolic differences
Culture characteristicsCulture characteristics– MacConkey agarMacConkey agar is both is both
selective and differentialselective and differentialBile salts and dyes inhibit Bile salts and dyes inhibit all but certain gram-all but certain gram-negative rodsnegative rods
– ““SelectiveSelective””
Acid produced by bacteria able to ferment lactose Acid produced by bacteria able to ferment lactose will turn a pH indicator red and form red colonieswill turn a pH indicator red and form red colonies
– ““DifferentialDifferential””
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
Metabolic differencesMetabolic differences
Biochemical testsBiochemical tests– Generally necessary for more conclusive Generally necessary for more conclusive
identificationidentification– Most rely on pH indicator or color change Most rely on pH indicator or color change
when a compound is degradedwhen a compound is degraded
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
Metabolic differencesMetabolic differencesBiochemical testsBiochemical tests– Sugar fermentationSugar fermentation
e.g., Lactose, sucrose, e.g., Lactose, sucrose, glucose, etc.glucose, etc.Fermentation results in Fermentation results in acid productionacid production
– pH indicator changes colorpH indicator changes color– Pink Pink yellow yellow
Inverted tube (Durham tube) collects any gas Inverted tube (Durham tube) collects any gas producedproduced
PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS
SerologySerologyProteins and polysaccharides of some Proteins and polysaccharides of some bacteria can function as identifying markersbacteria can function as identifying markers– Generally molecules on surface structuresGenerally molecules on surface structures
e.g., Cell wall, glycocalyx, flagella, pilie.g., Cell wall, glycocalyx, flagella, pili
– Detection is based upon the Detection is based upon the specific interaction between specific interaction between antibodiesantibodies and these and these antigensantigens
e.g., Rapid detection of e.g., Rapid detection of Streptococcus pyogenesStreptococcus pyogenes
Hippuricase gene Hippuricase gene (HipO)(HipO)
HipOHipO code for hippuricase enzyme. code for hippuricase enzyme.
Catalyses the hydrolysis of N-benzoyleglycin Catalyses the hydrolysis of N-benzoyleglycin (Hippuric acid) to glycine and benzoic acid.(Hippuric acid) to glycine and benzoic acid.
HipOHipO gene present only in gene present only in C. jejuni C. jejuni but not in any but not in any other other Campylobacter Campylobacter spp.spp.
Hippuricase detection using the iCyclerHippuricase detection using the iCycler
C .jejuni
C. coli
C. hyointestinalis
C. upsaliansis
C. upsaliansis
C. hyointestinalis
C. coli
C. jejuni
292 bpPrimer dimer
1 2 3 4