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

PCR

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

Xifen

No. 3

nega

tive

cont

rol

Dongn

ong

No.

704

Shenf

en N

o. 2

Mao

hong

No.

1

Jifa

n No.

4Za

okua

iLi

chun

Zaof

enZh

ongs

u No.

5

Hongz

a No.

10

R144

Nongy

ou N

o. 3

01

Shuan

gfu

Shenn

ong

No. 2

Jiaf

en N

o. 1

Yashu

No.

6

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

“IDENTITY CARD”for Foodfor Animal Feed

Edited by Prof. Dr. Sudjadi

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 38

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

4343

Microorganism Growth in FoodsMicroorganism Growth in Foods

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