Item 7. SAMPLING AND LMOs
DETECTION
7.3. IDENTIFICATION OF LMOs
Detection is required when:
• By law in the country is required
identification and/or labelling
• Mixtures between GMOs + non-GMOs
• Need to export to a country with strict
legislation
• Need to verify non-GMOs shipments
• For environmental risk verifications
In the international market
• Important focus
differences between
commercial blocks:
– USA does not require
identification and GM
crops are easily set free
into the environment
– EU requires labelling with
0.9% treshold
“Historical” cans of GM tomato puree with long shelf life
• First GM-food authorized 1994
• They were labelled… and people bought them!!!
• The variety lost sensory characteristics and was retired from
the market
Extraction
of:
Sampling
Immunostrips
ELISA plates
Protein
PCR end point
DNA
RTq-PCR
In order to detect proteins, specific antibodies are required. The
antibodies are proteins with quaternary structure.
Lateral flow test, IMMUNOSTRIP
Control line. Antibodies antiIgG adsorbed
Antibodies anti-antigen
conjugated with enzyme
Test line. Antibodies antiantigen adsorbed
Lateral Flow test,
IMMUNOSTRIP
The antibodies are binded to their antigen in the
sample and the complex antigen-antibody moves by
capillarity towards the reaction lines.
Lateral Flow test,
IMMUNOSTRIP
Antigen binds to the
antibodies that are in the test
line.
Free antibodies bind to the
antibodies anti-Ig present in the
control line.
Immunostrip procedure
1. Weigh 250 mg of
fresh leaf (plantlet)
2. Insert the sample
in the bag
3. Grind or crush
the sample
5. Let stand for 10
minutes (vertical
position)
4. Insert the immunostrip
into the bag with SEB or
MEB buffer.
6. Read results
Qualitative immunoassay in strip.
Ej. Cry9C QuickStixTM Envirologix
Also by this technique can be detected CP4-EPSPS
protein in samples with low % of transgenic maize
IMMUNOSTRIP CP4 EPSPS
IMMUNOSTRIP Cry
1Ab/Ac
Maíz NK603
Maíz BT 11
10%
1%
0.1%
0.05
%
10%
1%
0.5
%
0.1
%
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Format: DAS (Double Antibody Sandwich)
The first Ac is adsorbed to
the plate
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Formato: DAS (Double Antibody Sandwich)
Sample that contains
protein is added
Protein
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Format: DAS (Double Antibody Sandwich)
Second antibody is added. It is
conjugated with a enzyme
Enzyme
Antibody
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Formato: DAS (Double Antibody Sandwich)
Colorless
compound
Product
Colorless
Product
compound
Enzyme
Antibody
ELISA plate, second antibody congujated to
alkaline phospatase
Quantitative Immunoassay (ELISA)
Centrifugation to clarify samples
Sample extraction.
Preparation of ELISA plates
ELISA cont.
Incubation at room temperature
Sample application
Wash
Substrate addition/
Colour development
Spectrophotometric
measurement
- for quantification-
Specificity assesment of antibodies
in the ELISA test
1
2
3
A
MEB
Cry 1Ab/1Ac NK 603
B
MEB
Cry 1Ab/1Ac NK 603
C
CP4
Cry 3A
Bt 11
D
CP4
Cry 3A
Bt 11
E
NPT II
Cry 3Bb1
MON 810
F
NPT II
Cry 3Bb1
MON 810
G
Cry 2A
Cry 1C
Chalqueño
H
Cry 2A
Cry 1C
Chalqueño
KIT Cry 1Ab/1Ac
KIT CP4 EPSPS
1 2 3
1 2 3
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
Quantitative Analysis by ELISA
in detection of protein CP4-EPSPS
1
1
2
3
4
5
A
A
MEB
0.125 %
B
MEB
0.125 % Mon810 10%
Nk603 10% Bt11 10%
C
1%
0.062 % Mon810 1%
Nk603 1% Bt11
1%
C
D
1%
0.062 % Mon810 1%
Nk603 1% Bt11
1%
D
E
0.5%
0.031 % Mon810 0.1% Nk603 0.1% Bt11 0.1%
F
0.5%
G
H
Mon810 10% Nk603 10% Bt11 10%
0.031 % Mon810 0.1% Nk603 0.1% Bt11 0.1%
Control
0.25% 0.015 % Chalqueño
MEB
Negativo
Control
0.25% 0.015 % Chalqueño
MEB
Negativo
B
E
F
G
H
2
3
4
5
Detection thresholds of three transgenic maize events
CP4-EPSPS protein can be detected with high sensitivity in mixtures with low
percentage of transgenic maize.
Sensitivity for detection of CRY proteins is much lower.
Considerations:
Immunochemical methods
• Immunochemical methods can be realized in
fast ways, in situ, or in laboratory, few
equipment is required.
• Strip based methods are only qualitative.
• ELISA can be quantitative but:
– Different levels of protein expression are reflected in
different sensitivity levels
– No reference materials recognized.
– No agreement between quantification units (% in
weight or protein concentration)
PCR principles
 Double chain opening (~90°C) –denaturation Primers that recognize specific sequences
(50- 60°C)
 Synthesis of template complementary
chains (74°C)
3’
5’
5’
3’
Sequence of interest
3’
5’
5’
Synthesis of one copy
3’
Taq polymerase
Amplification:
Sequence of interest
Taq
Taq
Amplification
Taq
Taq
Number of
PCR cycles
0
1
2
3
4
5
6
7
8
9
10
20
30
40
Number of DNA
molecules
1
2
4
8
16
32
64
128
256
512
1024
1,000,000
1,000,000,000
1,000,000,000,000
Theoretical
PCR
products
Real
Cycle number
Factors to consider
• Specificity – primer design
• Product length (DNA amplified fragment)
• There are differences between qualitative
and quantitative tests
• Whether PCR is uniplex or multiplex
• If the method is specific for a type of
instrument
Primer design
P
E
G
1. Exploration
2. Gene specific target
3. Specific
construct
4. Event specific target
H
P
E
G
T
Host genomic DNA
Promoter element (CaMV 35S)
Amplifying element
Gene of interest (Cry, EPSPS)
Terminator (NOS)
T
H
LOW
Target specificity
H
HIGH
PCR for transgenic
sequence detection
Recombinant Gene
1
A
2
3
4
B
5
C
Amplicons
D
DNA of GM grains
Genomic DNA
Recombinant gene
Specific-specie
primers recognize
genomic DNA
DNA of non-GM
grains
Genomic DNA
Specific-GMO
primers do not
interact
No amplification
Intrinsic factors of the sample that
affect the amplification
• Integrity of template DNA
– Size of the amplicon
• Presence of inhibitory substances
– Humic substances
– Proteins
Others:
– EDTA
– NaOH
– SDS and other detergents
A
1
2
3
4
5
6
7
8
2000 bp
1000 bp
1 Ladder
2A Seeds
B
1500 bp
500 bp
Load: 100ng
Dye: SYBR Green
2B Commercial
nixtamal flour
1
2
3
4
5
6
7
8
3
4
5
6
7
8
Dough
Nixtamal flour
Tortilla
Tortilla chips
Corn chips
Dry Corn chips
DNA Extraction
• Extraction yield
– Tissue
• DNA purity
• Quality for amplification
Amplification – effect of the purity
of the DNA template
A
M
S
E
HJ
HJS
HM
HMS
Es
P
C-
225
pb
C
C-
M
HM HMS
HJ
HJS
S
E
P
B
C-
M
HM
HMS
HJ
HJS
S
E
Es
P
225
pb
225
pb
Amplification of a fragment of the
Invertase gene.
Limit of Detection
MON810
B
M
10
1
0.1
C-1
C-2
C-3
C-4
300 pb
100 pb
Detection of the specific event MON810 in different proportions (10%, 1% y 0.1%). C-1, negative
control with BT11 maize seed DNA 100% transgenic; C-2, negative control with NK603 maize seed
genomic DNA; C-3, negative control with chalqueño maize seed DNA; C-4, negative control without DNA.
M, 50 bp ladder.
Identity verification:
Restriction analysis
Amplicons of CamV35S promoter and restriction products with Asp700.
Lane 1: 50bp ladder, lanes 2 and 3: Bt176 control, lanes 4 and 5: canned
corn grains, in 2% agarose gel.
Quantitative PCR
qPCR – RTQ-PCR
PCR
products
100
10
2
1
0.1
0.01
Cycle number
Ct
m=-3.32
Log conc.
Ct = number of cycles needed
for the amplification signal to
be statistically different from
the background signal
Number of
PCR cycles
Number of DNA
molecules
1
2
3
2
4
8
21
22
23
10
2 3.32
16
32
64
128
24
...
...
...
4
5
6
7
Lineal dynamic range
Efficiency
(-1/m)
Efficiency = ([10
]-1) * 100
Results are accepted when the efficiency
is higher than 95% (m = 3.45 a 3.3)
More common system probes
Some results
in PCR real time
Specificity of primers and probes designed for RTQ-PCR
Maíze
%
GM
O
Primers and Probes
Bt11
Primers and Probe
MON810
Primers and Probe
Promoter 35S CaMV
Primers and Probe
Endogenous gene
No. muestras/
reacciones
positivas
Promedi
o
de Ct
No. muestras/
reacciones
positivas
Promedio
de Ct
No. muestras/
reacciones
positivas
Promedio
de Ct
No. muestras/
reacciones
positivas
Promedio
de Ct
Bt11
10
3/3
29.2+0.1
0/3
-
8/8
26.1+0.2
10/10
23+0.33
MON810
10
0/3
37.9 *
6/6
27.4+0.1
5/5
26.4+0.1
10/10
22.9+0.5
NK603
10
0/3
39.8+0.5
*
0/3
-
4/4
25.7+0.2
6/6
23+0.16
Chalqueño
0
0/3
-
0/3
-
0/6
35.9+0.3*
10/10
23+0.42
NTC
-
0/3
-
0/3
-
0/4
-
0/10
-
Effect of the extraction system
Figure 26. Standard curves of the events MON810 y Bt11 from DNA extracted with the commercial systems A, B and
C. 1, curve of the event Bt11 with extraction system A. 2, curve of the event MON810 with extraction system A. 3,
curve of the event MON810 with extraction system B. 4, curve of the event Bt11 with extraction system B. 5, curve of
the event Bt11 with extraction system C. 6, curve of the event MON810 with extraction system C.
A
B C
D
E
F G H
Figure 27. Amplification curves generated with 100% transgenic DNA
from event MON810, extracted with the system B. A, 20 ng. B, 10 ng.
C, 5 ng. D, 2.5 ng. E, 1.25 ng. F, 0.625 ng. G, 0.313 ng. H, 0.156 ng.
Figure 29. Amplification curves generated with 100% transgenic DNA of the
event MON810, extracted with the system A. Serial dilutions were performed
however, the amplification generated with each dilution does not allow to
clearly establish to each curve the initial DNA concentration.
Effect of the extraction method
over the quantification
Mixture of MON810 (%)
B
Extraction
method
C
A
10
1
0.1
11.48 + 0.327
0.91 + 0.186
0.11 + 0.032
16.15 + 1.118
2.11 + 0.154
0.12 + 0.016
13.96 + 0.140
Without
amplification
Without
amplification
Effect of the primers and
probes design
Linearization Data
Kit
comercial
OGMs
Transgene
Diseño
Endogenous
Gene
Kit
comercial
OGMs
Diseño
Curve
with event
Slope
Intercept
R2
Efficiency
(%)
Bt11
-3.537
28.253
0.999
91.7
MON810
-3.375
29.712
0.999
97.8
NK603
-3.836
30.512
0.999
82.3
Bt11
-3.382
27.673
0.998
97.5
MON810
-3.326
28.411
0.998
99.8
NK603
-3.429
29.592
0.992
95.7
Bt11
-3.685
29.003
0.993
86.8
MON810
-3.516
29.466
0.996
92.5
NK603
-3.731
30.187
0.985
85.4
Bt11
-3.111
28.593
0.997
109.6
MON810
-3.326
28.228
0.995
99.8
NK603
-3.193
29.103
0.995
105.7
Sybr green
A
B
First negative derivate of the
dissociation curves of
Amplicons obtained for
A. MON810 and
B. endogenous gene
Non-specific amplification
Effect of the DNA quality over
Sybr green quantification
Mixture of MON810 (%)
Extraction
method
10
1
0.1
B
10.3±0.007
1.1±0.021
0.25±0.006
C
10.8±0.018
2.3±0.017
0.10±0.077
A
48.5±0.157
53.8±0.149
0.03±0.011
Other example:
• Detection of transgenic maize in
processed foods:
– Nixtamal Flour
– Dough
– Tortilla
– Fried tortilla (tostada)
– Fried dough
M. Quirasco, B. Schoel, J. Plasencia, J. Fagan & A. Gálvez. 2004.
Suitability of RTQ-PCR and ELISA for Cry9C detection in Mexican corn
tortillas: fate of DNA and protein after alkaline cooking.
Journal of AOAC International. 87:639-646
A
1
2
3
4
5
6
7
8
2000 bp
1000 bp
1 Ladder
2A Semillas
B
1500 bp
500 bp
Carga: 100ng
Tinción: SYBR Green
1
2
3
4
5
6
7
8
2B Harina de
nixtamal comercial
3 Masa
4 Harina de nixtamal
5 Tortilla
6 Tortilla frita
7 Masa frita
8 Masa seca frita
Content of GMO, determined by RTQ-PCR, in different nixtamalized products prepared with
white maize non – transgenic and different percentages of StarLinkTM .
StarLinkTM 0.1% (w/w)
Sample
Granos de
maíz
Masa
Media, %
(w/w)
0.19
RSD
CV, %
0.018
9.3
0.10
0.005
4.8
Harina de
nixtamal
Tortilla
0.10
0.001
0.7
0.31
0.004
1.4
Tortilla
chip
Corn chip
ND
-
-
0.10
0.002
1.5
Corn chip
secos
ND
-
ND = No detectado
StarLinkTM 1% (w/w)
Sample
RSD
CV, %
0.121
9.8
1.16
0.002
0.2
Harina de
nixtamal
Tortilla
1.03
0.016
1.5
1.41
0.015
1.0
Tortilla
chip
Corn chip
0.52
0.001
0.3
1.16
0.023
2.0
0.079
12.4
Granos de
maíz
Masa
Corn chip
secos
Media , %
(w/w)
1.23
0.63
StarLinkTM 10% (w/w)
Sample
RSD
CV, %
0.166
1.2
12.64
0.090
0.7
Harina de
nixtamal
Tortilla
9.35
0.017
0.2
9.47
0.254
2.7
Tortilla
chip
Corn chip
6.64
0.175
2.6
14.29
0.119
0.8
0.053
0.6
Granos de
maíz
Masa
Corn chip
secos
Media, %
(w/w)
14.12
8.28
LOD = Limit of Detection
=.01% en RTq-PCR
LOQ = Limit of Quantification
= 0.1% en RTq-PCR
Immunochemical methods are adequate to detect the protein in
grains and materials without too much processing
Protein
DNA
Seed
Primary
Processed
ingredient
foods
Highly processed
foods
... However DNA can be detected in highly processed foods
Sampling
Pre-treatment
GenScan
Halving 1
Halving 2
Composite
samples
“
1
2
UNAM
3
Detection and
cuantification of
exogenous DNA by
RTQ-PCR
ELISA
p35S
Detection of authorized
and unauthorized
transgenic events in
Mexico by RTQ-PCR
Heterologous
protein
detection
Event
Specific
Immunostrips
Results
Cd. Juárez
Piedras Negras
Nuevo Laredo
Matamoros
Altamira
Customs
where
maize samples
were obtained
Veracruz 430
Veracruz Centro
Coatzacoalcos
1)
2)
3)
4)
Subsampling by dividing into four parts (“halving”)
Food authorization status of
GM varieties of maize
TRANSFORMATION EVENT
Authorized in
Mexico
U.S.A.
Europe
MON-00021-9 (GA21)
Yes
Yes
Yes
MON-00603-6 (NK603)
Yes
Yes
Yes
MON-00810-6 (MON810)
Yes
Yes
Yes
DAS-01507-1 (TC1507)
Yes
Yes
Yes
MON-00863-5 (MON863)
Yes
Yes
Yes
DAS-59122-7
Yes
Yes
Yes
MON-88017-3 (MON88017)
Yes
Yes
No
ACS–ZM002-1 / ACS-ZM003-2 (T14, T25)
Yes
Yes
Yes
SYN-BTØ11-1 (BT11 (X4334CBR, X4734CBR))
Yes *
Yes
Yes
REN-00038-3 (LY038)
Yes *
Yes
No
SYN-IR604-5 (MIR604)
Yes *
Yes
No
* vents Approved in october of 2007
Immunological methods commercially
available for heterologous protein detection
ELISA
• CP4-EPSPS (RR)
• Cry3Bb1
• Cry1Ab/1Ac
• Cry1F
Immunostrips
• CP4-EPSPS (RR)
• Cry3Bb1
• Cry1Ab/1Ac
• Cry1F
• Cry34Ab1
• Cry9C
• PAT
Possible presence of transgenic events by immunoassay
(ELISA + strips)
Protein
Port 1
Port 2
Port 3
Poprt 4
Port 5
Port 6
Port 7
Port 8
Cry1Ab/Ac
MON810
Bt11
MON80100*
MON802*
MON809*
MON810
Bt11
MON80100*
MON802*
MON809*
MON810
Bt11
MON80100*
MON802*
MON809*
DBT418**
MON810
Bt11
MON80100*
MON802*
MON809*
MON810
Bt11
MON80100*
MON802*
MON809*
DBT418**
MON810
Bt11
MON80100*
MON802*
MON809*
DBT418 **
MON810
Bt11
MON80100*
MON802*
MON809*
MON810
Bt11
MON80100*
MON802*
MON809*
Cry3Bb1
MON88017
MON863
MON88017
MON863
MON88017
MON863
MON88017
MON863
MON88017
MON863
MON88017
MON863
MON88017
MON863
N/D
Cry1F
DAS01507
DAS01507
DAS01507
DAS06275 ***
DAS01507
DAS01507
DAS06275***
DAS01507
DAS06275 ***
DAS01507
DAS01507
Cry34Ab1
DAS59122
DAS59122
DAS59122
DAS59122
DAS59122
DAS59122
DAS59122
ND
ND
ND
ND
ND
ND
ND
ND
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
NK603
MON88017
MON80100*
MON802*
MON809*
ND
MON863
DAS59122
T14/T25
DAS06275***
DBT418**
MS6
ND
MON863
DAS59122
T14/T25
DAS-06275***
DBT418**
MS6
MON863
DAS59122
T14/T25
DAS06275***
DBT418**
MS6
ND
ND
Cry9C
CP4EPSP
S
PAT†
ND
ND, non detected protein
* Events not containing Cry1Ab/Ac + CP4EPSPS
** Event containing Cry1Ab/Ac + PAT
*** Event containing Cry1F + PAT
† StarLink contains Cry9C + PAT. Cry9C not detected, its presence is discarded in PAT
positive samples
Potential presence of MON80100 y MON809 because they were not commercialized. DNA
verification required.
Specific presence of transgenic events in maize grains,
by RTQ-PCR
Event
MON810








T25








GA21








NK603








DAS-01507-1








MON863








MON88017








DAS-59122-7








Third party analysis (Gene Scan)
• Confirmed the results obtained in Lab. 312, Dept.
of Food Science and Biotecnhnology, Faculty of
Chemistry, UNAM
• Confirmed the absence of the eventMON88017
“Possible” events are discarded
• Immunodetection is insufficient for specific-event
detection.
• Do not encompass the totality of
authorized events (GA21), as well as nonauthorized ones (676/678/680, DLL25,
LY038, MIR604, MS3 y MS6)
• By PCR was confirmed the presence of MON810, MON862,
DAS01507, DAS59122 and NK603
• It was also discarded the presence of MON88017, possible event
according to the immunodetection.
• Technological development is needed
for the detection of possible unauthorized events.
60
50
40
%
30
20
10
0
Events
Customs
Quantification (RTQ-PCR) of transgenic events authorized in
Mexico prior to may 2007*
* Bt11 y LY038 approved in july andy MIR604 in october of 2007
140
120
100
%
80
60
40
20
0
C
Aduana
ua
ah
u
hi h
i la
hu
a
Co
as
l ip
u
ma
Ta
Estado
uz
rac
e
V
e
o rt
N
a
n
Fro
ter
d
lfo
o
G
o
ic
éx
M
e
Región
Total content (%) of GM material in each one of the
analyzed samples
“Stacked” varieties of Maize
Event 1
x
Event 2
ACS-ZMØØ3-2
x
MON-ØØ81Ø-6
DAS-59122-7
x
NK603
DAS-59122-7
x
TC1507
DAS-Ø15Ø7-1
x
MON-ØØ6Ø3-6
BT11
x
MIR604
BT11
x
MIR604
MON-ØØ6Ø3-6
x
MON-ØØ81Ø-6
MON-ØØ81Ø-6
x
LY038
MON-ØØ863-5
x
MON-ØØ6Ø3-6
MON-ØØ863-5
x
MON-ØØ81Ø-6
MON-ØØ863-5
x
MON-ØØ81Ø-6
MON-ØØØ21-9
x
MON-ØØ81Ø-6
MON89034
x
MON88017
MON8903
x
NK603
SYN-BTØ11-1
x
MON-ØØØ21-9
TC1507
x
DAS-59122-7
MIR604
x
GA21
MON810
x
MON88017
x
Event 3
x
NK603
x
GA21
x
MON-ØØ6Ø3-6
Copia 1 ADN
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN
Evento “Stack”
DAS59122-7
x
100%
NK603
100%
200% ?
Copia 1 ADN
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN
Evento “Stack”
DAS59122-7
x
80%
NK603
100%
180% ?
Copia 1 ADN
(No-GM)
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN
Evento “Stack”
DAS59122-7
x
80%
NK603
80%
160% ?
Normalizing genes from
developers´ certified methods*
• MON810: hmg (high mobility group)
• MON863: adh (alcohol dehydrogenase)
• NK603: adh (alcohol dehydrogenase)
* Only recomended gene
Use of normalizing gene in tests performed
in the laboratory: RT-PCR
• % OGM = (transgene/normalizing gene) x 100
• MON810 with hmg
• MON810 with adh
15.25 x 100 = 43.8
34.84
15.25 x 100 = 23.1
66.01
• If the amount of endogenous gene increases (denominator),
the amount of GMO will be underestimated.
90
80
70
1
2
60
3
%
50
4
40
5
6
30
7
20
8
10
0
hmg
adh
MON810
hmg
adh
MON863
hmg
adh
NK603
Quantification of three events using
two different normalizing
• adh could be present in the genome of the maize in
more than one copy.
• Quantification with adh could underestimate the
results
140
120
100
Eventos
80
p35S
%
60
40
20
8
7
6
5
4
3
2
1
0
Quantification of GM material (total) according
to specific events or promoter 35S
Hipotesis about the diferent quantification
of the endogenous gene
• Poor characterization of the endogenous gene (number
of copies in the genomic DNA not specified)
• Sequences of primers or probes provided by the
developer are not specific enough
• Low astringency PCR programme
• ¿Which endogenous gene should choose?
• ¿Differences between cultivars?
Conclusions
• Exists a high income of GM material in the analyzed customs.
• The most abundant events were: MON810 > NK603 >
DAS1507-1
• The presence of “stacked” variety makes more complex the
transgenic material quantification.
• Standardization is required (at international level) regarding
which normalizing genes should be used.
• An inter- governmental information exchange system is
required in order to harmonize maize commerce between the
USA and Mexico
• The event MON88017 (authorized variety) was not detected
Abraham Acatzi1
Javier Magaña1
Carlos Moles2
Carolina Peña2
Marcela Castillo2
Maricarmen Quirasco1
Javier Plasencia3
Marcelo Signorini4
Amanda Gálvez1, 2
1
Programa Universitario de Alimentos. PUAL-UNAM
Dept. Alimentos y Biotecnología. Facultad de Química. Universidad Nacional Autónoma de
México (UNAM)
3 Dept. Bioquímica. Facultad de Química. UNAM
4 Comisión Federal para la Protección contra Riesgos Sanitarios. Secretaría de SALUD
2