Date post: | 14-Jun-2015 |
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• Authors: Fa´ bio Cristiano Angonesi Brod, Cibele dos Santos Ferrari, Luciana Lehmkuhl Valente, Ana Carolina Maisonnave Aris
(Universidade Federal de Santa Catarina, Brazil)
By Khang DT
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I. INTRODUCTIONI. INTRODUCTION
II. MATERIAL AND METHODII. MATERIAL AND METHOD
III. RESULTS AND DISCUSSIONIII. RESULTS AND DISCUSSION
IV. CONCLUSIONIV. CONCLUSION
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• Genetically modified orgamism (GMO)
Multifunction Productivity
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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• The herbicide-tolerant Roundup ReadyTM (RR)
Soybean (Monsanto) in Brazil
Herbicide tolerant soybean
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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• Article 21 For commerce of foodstuffs and food ingredients, destined for human or animal consumption, containing GMO above the 1% threshold based on product level, the consumer must be informed of the transgenic origin of this product.’
• Labeling requirement
HOW TO DETECT GMO???
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
Polymerase chain reaction (PCR)
Kary Mullis accepting the Nobel Prize PCR amplification
in 1993
Nested PCR increases the confident level
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MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
DNA
Nested PCR region
PCR region
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1. Samples1. Samples
The soy products (six defatted soy flours, six infant formulas containing 14% soy protein isolate and 25 powdered soymilks) were purchased from local supermarkets and pharmacies in Floriano´ polis,Brazil.
2. DNA extraction2. DNA extraction
CTAB protocol
3. PCR conditions3. PCR conditions
4. Agarose gel electrophoresis4. Agarose gel electrophoresis
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
2. PCR protocol2. PCR protocol• 1XPCR buffer (20 mmol/l Tris-HCl, pH 8.4, 50 mmol/l KCl),
• 2.5 mmol/l MgCl2,
• 0.2 mmol/l of each dNTP, • 0.5 mM of each primer, • 1 unit of Taq DNA polymerase (Invitrogen)• 2 ml of DNA (maximum 50 ng)
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PCR CONDITIONSPCR CONDITIONS
95o
12:00m0:30s
95o
62o
72o
1:00m
0:30s∞
4o
Temperature
Time
50 cycles1. PCR thermal cycle1. PCR thermal cycle
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3. PCR primers3. PCR primers
Primer Sequence (50-30) Amplicon size (bp)
LEC1 GTGCTACTGACCAAGGCAAACTCAGCA 164 LEC2 GAGGGTTTTGGGGTGCCGTTTTCGTCAAC
GMO09 CATGAAGGACCGGTGGGAGAT 447GMO05 CCACTGACGTAAGGGATGACG
GMO08 TGGGGTTTATATGGAAATTGGAA 169GMO07 ATCCCACTATCCTTCGCAAGA
Primers GMO5 and GMO7 are complementary to the CaMV 35S promoterPrimers GMO9 hybridizes to the CP4 EPSPS sequence and GMO8to the CTP sequence
GMO8 GMO7 GMO5
GMO9
CaMV35S promoter CTPCP4 EPSPS
DNA
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1. Samples1. Samples
The soy products (six defatted soy flours, six infant formulas containing 14% soy protein isolate and 25 powdered soymilks) were purchased from local supermarkets and pharmacies in Floriano´ polis,Brazil.
2. DNA extraction2. DNA extraction
CTAB protocol
3. PCR conditions3. PCR conditions
4. Agarose gel electrophoresis4. Agarose gel electrophoresis
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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Fig. 1. Amplification of soybean lectin gene.
Lane 1: 50 bp ladder (Promega); lane 2: negative control (water); lane 3: positive control (soybean DNA); lanes 4–14: powder soymilks.
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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Fig. 2. RR soybean detection by nested PCR.
Lane 1: 50 bp ladder (Promega); lane 2: positive control (soybean 0.1% RR); lane 3: negative control (water); lane 4: soybean 0% RR; lane 5: soybean 0.001% RR; lane 6: soybean 0.01% RR; lane 7: soybean 0.1% RR; lane 8: soybean 1% RR; lane 9: soybean 10% RR (8 ml PCR product+2ml loading buffer per lane).
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
All soybean mixed samples containing 0.01–10% GM soybean.This fragment was absent for 0% GM soybean andalso for 0.001% GM soybean
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Fig. 3. RR soybean detection by nested PCR.
Lane 1: 50 bp ladder (Invitrogen); lane 2: negative control (water); lane 3: negative control (soybean 0% RR); lane 4: positive control (soybean 10% RR); lanes 5–10: soy flours, first DNA extraction; lanes 11–16: soy flours, second DNA extraction.
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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Fig. 4. RR soybean detection by nested PCR.
Lane 1: 50 bp ladder (Invitrogen); lane 2: negative control (water); lane 3: positive control (soybean 0.1% RR); lane 4: negative control (soybean 0% RR); lanes 5–15: powder soymilks
MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
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MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
• In six analysed soy flour samples, four samples were positive for the presence of RR soybean
and in 25 analysed soymilk powder samples, 15 showed a positive signal of 169 bp length for the nested PCR detection of RR soybean.
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MATERIAL & MATERIAL & METHODMETHOD
MATERIAL & MATERIAL & METHODMETHOD
RESUL & RESUL & DISCUSSIONDISCUSSION
RESUL & RESUL & DISCUSSIONDISCUSSION CONCLUSIONCONCLUSIONCONCLUSIONCONCLUSIONINTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION
This study shows that the nested PCR method developed by Meyer and Jaccaud (1997) is adequate to determine the presence of GM soybean in samples of soybean flour, soymilk powder and infant formula containing protein isolate.
This nested PCR method could be employed to distinguish GM from nonGM products, because the legal requirement for the labeling of foods containing GMO.
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