Bayh-Dole: Background and Implementation

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
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 yellowishorange color, thus, the name ‘Golden Rice’
Building the Transgenes
ON/OFF Switch
PROMOTER
Makes Protein
CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
stop sign
poly A signal
Examples of engineered species
Wheat (Triticum aestivum) - a man-made
species
Corn (Zea mays) - derived from teosinte
Soybeans (Glycine max) - from G. soja
Potatoes (Solanum tuberosum) - wild
varieties 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)
x
x
x
Transgenic
line
Commercial
variety
Commercial Transgenic Line
Biotechnology
Advantages of GM Foods
1. Pest Resistance
2. Herbicide Tolerance
3. Disease Resistance
4. Drought Tolerance
5. Cold Tolerance
6. Nutrition
7. Pharmaceuticals
8. Phytomediation
9. Enhanced taste and quality
10. Reduced maturation time
Disadvantages of GM Foods
1. Environmental Hazards
2. Economical Issues
3. Human Health Risks
– Causes humans to possibly become more
allergic to GM goods
– Unknown effects on human health
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
Type of Policy
GM Content Threshold
Labelling
Policies
Countries/Regions
Australia/New Zealand
China
Czech
European Union/UK
Hong Kong
Japan
Russia
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Switzerland
Mandatory
Brazil (draft)
India (draft)
Israel (draft)
Malaysia (draft)
Korea (draft)
Taiwan (draft)
Thailand (draft)
Canada
USA
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Mandatory
Voluntary
Voluntary
1%
n/a
1%
0.9%
5%
5%
5%
2% or 3% depending on
different situations
n/a
n/a
1%
3%
3%
5%
5%
5%
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)
GM products should be labeled
Soybean
Corn
seed, soybean, flour, oil, soy meal
seed, corn, flour, oil, corn meal
Rapeseed
seed, rapeseed, oil, rapeseed meal
Tomato
seed, 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
procedure
Samples
Sampling
Tested Samples
Protein Detection
Methods
ELISA
Lateral Flow Strip
Saved Samples
Nucleic Acids
Detection Methods
DNA Extraction
Conventional PCR
Negative
No GM contents
Positive
Contained
GM contents
Quantitative PCR
GM Contents (xx%)
PCR
Levels of specificity – GM targets (DNA)
plant genomic DNApromoter
gene
terminator plant genomic DN
Screening targets
LOW
Gene specific targets
Construct specific
Event specific targets
HIGH
Arne 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 AcUNIF/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 endogenous reference gene: Lat52
Allelic variation analysis
Southern blot analysis
confirmed that this gene was
single copy in the tested
varieties
Tomato seeds Ketchup tomato juice
Journal of Agriculture Food Chemistry 2005,50(2):122-125
Screen and construct specific detection of
transgenic Huafan No.1 tomato
CaMV35s
promoter
A
AntiEFE
NOS
terminator
EcoR I
Hin
d
III
195bp
PCaMV35S
180bp
Nos
Terminator
B
C
Huafan construct
153bp specific fragment
Journal of the Science of Food and Agriculture 85:2159–2166 (2005)
Event-specific PCR Detection of Genetically Modified
Soybean GTS 40-3-2
M
1
2
3
4
5
6
7
8
Conc= 10^(-0.323*CT + 12.302)
R^2＝0.9993
Specific analysis
M
1
2
3
4
5
6
7
8
9
LOD:2 copies，LOQ: 20 copies。
Standard deviation repeatability and
Sensitivity analysis
reproducibility were less than 0.2
Coefficient Values of the reference molecule was 0.92 sample analysis
Event-specific PCR detection of two GM-rapeseed （RT73 and T45 ）
Specific analysis
LOD and LOQ in event-specific quantitative
PCR detection of event T45 were 1 copy and
10 copies respectively;For RT73, LOD and
LOQ were 10 copies and 100 copies
respectively。
A
。
B
Sensitivity analysis
（A）T45；（B）RT73
B
A
B
Based on the standard curve , rapeseed
samples were analysised by quantitative
PCR.
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
“IDENTITY CARD”
for Food
for Animal Feed
Edited by Prof. Dr. Sudjadi
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 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.
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.
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
….and traceability
Panic Over Smuggled Meat
Grows
27 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.
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.
Thursday, 21 December, 2000, 15:27 GMT MP urges
action on meat fraud
The appeal came after the conviction of five people for a multimillion pound meat fraud uncovered by environmental health
officers in Rotherham.
 Traceability is never 100% ensured
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
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
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
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
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)
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)
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’
International Food Research Journal 18(4): 1489-1491 (2011)
Modifyed by Sismindari
29/7/2011
37
29/7/2011
Modifyed by Sismindari
38
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
(1) DNA marker, (2) Beef meatball (MB),
(2) (3) Pork Meatball
29/7/2011
Modifyed by Sismindari
39
Microorganism Growth in Foods
43
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 (nondiarrheal illness)
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
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
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
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
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
PHENOTYPIC CHARACTERISTICS
Metabolic differences
Culture characteristics
– MacConkey agar is both
selective and differential
Bile salts and dyes inhibit
all but certain gramnegative rods
– “Selective”
Acid produced by bacteria able to ferment lactose
will turn a pH indicator red and form red colonies
– “Differential”
PHENOTYPIC CHARACTERISTICS
Metabolic differences
Biochemical tests
– Generally necessary for more conclusive
identification
– Most rely on pH indicator or color change
when a compound is degraded
PHENOTYPIC CHARACTERISTICS
Metabolic differences
Biochemical tests
– Sugar fermentation
e.g., Lactose, sucrose,
glucose, etc.
Fermentation results in
acid production
– pH indicator changes color
– Pink  yellow
Inverted tube (Durham tube) collects any gas
produced
PHENOTYPIC CHARACTERISTICS
Serology
Proteins and polysaccharides of some
bacteria can function as identifying
markers
– Generally molecules on surface structures
e.g., Cell wall, glycocalyx, flagella, pili
– Detection is based upon the
specific interaction between
antibodies and these
antigens
e.g., Rapid detection of
Streptococcus pyogenes
Hippuricase gene
(HipO)
HipO code for hippuricase enzyme.
Catalyses the hydrolysis of N-benzoyleglycin
(Hippuric acid) to glycine and benzoic acid.
HipO gene present only in C. jejuni but not in any
other Campylobacter spp.
Hippuricase detection using the iCycler
C .jejuni
C. coli
C. hyointestinalis
C. upsaliansis
1
4
C. upsaliansis
C. hyointestinalis
C. coli
C. jejuni
2
3
292 bp
Primer
dimer