P-nos

Anhang 1
Real-time PCR method for the detection of the nopaline synthase promoter
(P-nos) and neomycin phosphotransferase gene (nptII) construct in genetically
modified crops
Draft of 14.10.2009
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1. Introduction and general information
The following method can be used to detect a transgenic construct present in several
genetically modified crops. The real-time Polymerase Chain Reaction (PCR) protocol uses
target-specific oligonucleotides that amplify a DNA fragment specific to the junction of a
transgenic construct of the nopaline-synthase gene promoter (pnos) from Agrobacterium
tumefaciens and the neomycin phosphotransferase II gene (nptII) from the Tn5 transposon of
Escherichia coli strain K12. The size of the amplified fragment is varying and depends on the
GM plant event. For detection of amplicons a FAM-labelled TaqMan probe binds to the nptII
part of the amplified sequence. The increase of the fluorescence signal is detected and
indicates the presence of the amplified pnos-nptII sequence.
The method has been developed by Dr. Ralf Reiting, Landesbetrieb Hessisches
Landeslabor, Standort Kassel, Druseltalstr. 67, 34131 Kassel / Germany and has been
validated in a pre-ringtrial within the framework of the German §64 LFGB working group
‘Development of methods for identifying foodstuffs produced by means of genetic
engineering techniques’. A collaborative validation study according to the IUPAC/ISO/AOAC
Protocol [1] is in preparation.
1.1 DNA-extraction
Depending on the sample material and the matrices the DNA extraction procedure applied
should yield DNA of sufficient quantity and purity to ensure that the influence of inhibitors is
minimized. Standard procedures according to ISO 21571 (Annex 1.1, 3 and 4) [3] are
recommended for use. Additional purification steps using gel-filtration columns may be of
help to purify the extracted DNA.
1.2 Real-time PCR system
The single-plex PCR assay uses standard cycling conditions and the TaqMan chemistry. It is
optimised for use in real-time PCR instruments for plastic vessels. The oligonucleotide probe
uses FAM as the fluorescent reporter dye and a BlackBerry Quencher (BBQ). An ABI7500
instrument was used during the in-house validation of the method.
1.3
Targeted gene sequence
A frequently used antibiotic-resistance gene used as marker in GM plants is the nptII gene
from the Tn5 transposon of Escherichia coli strain K12 which confers resistance to
aminoglycoside antibiotics such as kanamycin. In several GM plant events the inserted nptII
gene coding sequence has been combined with a promoter element originating from the
nopaline synthase gene (P-nos) from Agrobacterium tumefaciens. Genetically modified
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plants containing the construct of the nptII gene in combination with the P-nos promoter are
listed in Table 1.
Table 1. GM plant events containing the P-nos-nptII construct and length of amplicons. n.d. = not
determined; n. av. = no data available
GM plant event
containing the P-nos-nptII
MS1/RF1 (RF2)
Topas19/2
55-1 (Sunup)
T-120-7
EH92-527-1
FP967
Zeneca (TGT7-F)
New Leaf (e.g. RBMT15-101
351N
1345-4
RM3-3; RM3-4; RM3-6
Common plant
species name
rapeseed
rapeseed
papaya
sugarbeet
potato
flax
tomato
potato
tomato
tomato
chicory
length of
amplicon (bp)
152
144
170
144
163
165
n.d.
n.av.
n.av.
n.av.
n.av.
Reference
BATS
Report [4]
The real-time PCR assay amplifies the junction of the P-nos promoter sequence and the nptII
gene sequence. The fragment size generated by the P-nos-nptII PCR depends on the
assembly of the two sequence elements and is varying in the different GM plant events. The
consensus sequence of the P-nos-nptII construct and the location of the primers and the
probe as well of the variable region is illustrated in Figure 1.
TTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCANNNNNNNNNNNNNNGCAGGT
TCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATC
Figure 1: Consensus sequence of the P-nos-nptII construct present in the GM plant events as
listed in Table 1.The first 45 nucleotides originate from the P-nos promoter, while nucleotides 60 to
123 originate from the nptII gene. The variable sequence between the two elements is indicated by
15 N nucleotides. The location of the forward and reverse primers are underlined and given in bold
italics, the probe sequence is shown in bold and underlined letters.
The sequences for the design of oligonucleotide primers and the probe were derived from
GenBank accession number FJ467933.1 (complete coding sequence of the inserted
sequence in Carica papaya transgenic line 55-1). The forward primer matches to the nos
promoter sequence, while the reverse primer and the probe are complement to the 5’ part of
the nptII sequence.
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1.4
Reference materials
DNA of potato event EH92-527-1 (IRMM reference material ERM-BF 421b) was used as
reference material. In addition, DNA of GM rapeseed events Topas19/2 and MS1/RF1, of
GM papaya (Sunup) event 55-1 (positive market sample) and of GM flax event FP967
(received from LGL Oberschleißheim) was used as reference material. DNA extracted from
GM sugar beet event T120-7 was available from a field trial experiment in Hessen/Germany.
GM ‘Zeneca’ tomato (event TGT7-F) had been used in a ring trial of the German §64 LFGB
working group.
2. Real-time PCR
2.1
Reference gene specific PCR
In order to control the quality and quantity of the sample DNA a reference gene specific PCR
targeting a species-specific gene is recommended to be performed before or in parallel to the
P-nos nptII construct-specific PCR. The template DNA is analysed in different dilutions in
order to detect PCR inhibition. The Ct values measured for each of the dilutions should
correspond to the dilution factor, e. g. if the DNA is diluted four times, the measured Ct
difference should be approximately 2.
If a reference gene real-time PCR method is not available for the species of interest, a
universal plant DNA detection method may be used [4].
2.2
Oligonucleotide sequences
Sequences of primers and of the probe are given in Table 2.
Table 2. Sequences of Primers and the probe
1
Name
Oligonucleotide DNA Sequence (5’ to 3’)
p-nos-F2
TTC CCC TCG GTA TCC AAT TAG AG
NPTII-R
GAT TGT CTG TTG TGC CCA GTC A
NPTII-Tm2
FAM1 – AgC CgA ATA gCC TCT CCA CCC AAg C –BBQ2
FAM: 6-carboxyfluorescein; BBQ: Black Berry Quencher
2.3
Real-time PCR setup
The method is described for a total PCR volume of 25 µl per reaction with the reagents as
listed in Table 3.
For each sample DNA at least two reactions have to be performed.
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Table 3. Reaction mixture for detection of the P-nos-nptII construct
Component
Final
Concentration
Volume
per Reaction
(in µl)
Quantitect Multiplex PCR Mastermix (2x) No Rox*
1x
12,5
Primer p-nos-F2
400 nM
1
Primer NPTII-R
400 nM
1
Probe NPTII-Tm2
100 nM
0,25
Water (PCR quality)
__
5,25
Template DNA (200 ng genomic DNA)
__
5
Total volume
25
*other master mixes could be used if it is shown that they perform equally or better.
2.3
Cycling programme
The temperature-time programme for the P-nos-nptII real-time PCR as outlined in Table 4
was optimized for the ABI7500 real-time cycler. If other mastermixes are used, the time for
denaturation (step 1) may be adapted.
Table 4. Temperature-time programme (ABI 7500)
Step
Stage
Temperature
Time
(sec)
Data
Acquisition
Cycles
1
Denaturation
95 C°
900
NO
1
2
Amplification
94 C°
15
NO
60 C°
60
YES
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45
3.
In-house Validation
3.1
Specificity
The specificity of the P-nos - nptII screening method was tested against the several
genetically modified plants as given in Table 5. No amplification was observed for the GM
plant events listed.
Table 5. Specificity tests
Plant Species
GM Event
Result
Mon863; 59122; Bt11; Bt176; GA21; MIR604;
Corn
No amplification
Mon810; NK603; TC1507;
Rapeseed
Falcon GS49/90; GT73; MS8/RF3; Oxy235; 23198 (Laurical)
No amplification
Soy
GTS 40-3-2
No amplification
Rice
Bt63
No amplification
Sugar beet
GTSB77
No amplification
3.2
Correlation coefficient R2
In order to determine the performance of the P-nos - nptII method for the different targeted
GM plant events, dilutions of DNA from different p-Nos - nptII positive GM plant events were
prepared and analyzed in the real-time PCR assay. Five or six dilution steps (twofold or
fourfold dilutions) were analysed in duplicate reactions and the measured Ct values were
used to calculate the slope and the correlation coefficient R2. The results of these
experiments and the calculated slope and R2 values are listed in Table 6.
Table 6. Results obtained with dilution series of DNA from different GM plant events containing the
pnos-nptII gene construct
GM plant Event
Plant
Amplicon
Length (bp)
Measured Ctvalue range
R2 Coefficient
Slope
MS1/RF1
Rapeseed
152
24,9 - 35,1
0,998
-3,38
Topas19/2 –
Rapeseed
144
25,8 - 36,6
0,988
-3,52
55-1- (Sunup)
Papaya
170
28,7 - 33,7
0,991
-3,30
T-120-7-
Sugar beet
144
26,3 - 34,7
0,995
-3,35
EH92-527-1-
Potato
163
26,1 - 36,1
0,997
-3,22
FP967
Flax
165
28,2-33,4
0,991
-3,19
Zeneca TGT7-F
Tomato
n. d.
26,4-36,2
0,984
-3,31
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3.3
Limit of Detection (LOD)
The LOD was determined by the analysis of a dilution series of DNA from potato event
EH92-527-1. At a DNA dilution corresponding to 10 copies of the target sequence per
reaction 15 of 15 reactions resulted in positive amplification at an average Ct value of 36,4
±0,9.
In order to verify the LOD result with another GM plant containing the P-nos - nptII construct,
the LOD was additionally determined by the analysis of a dilution series of DNA from
rapeseed event Topas19/2. At a DNA dilution corresponding to 10 copies of the target
sequence 15 of 15 reactions resulted in amplification at an average Ct 36,7 ±0,8. The values
may differ in correspondence to the copy number of the construct present in the GM plant
event that is analysed.
3.4
Robustness
The robustness of the method has been evaluated in an interlaboratory comparison study
with four participating laboratories. The laboratories used different thermostable DNA
polymerases (ABI Taqman Universal Mastermix; Roche Probes Mastermix and Qiagen
Quantitect Mastermix) and four different real-time cyclers (ABI7900; ABI7900HT;
LightCycler480 and ABI7500). The results showed that the method is robust and transferable
to other laboratories without problems.
4.
Summary
The construct-specific P-nos – nptII real-time PCR method described here is proposed for
advanced screening purposes. The method uses primer and probe sequences targeting the
junction of the construct of the nos promoter (P-nos) from Agrobacterium tumefaciens and
the nptII from the Tn5 transposon of Escherichia coli strain K12 which is present in several
genetically modified plant events. The performance of the real-time-PCR assay was in-house
validated for seven different GM plant events. Even though the lengths of the PCR amplicon
differ slightly because of different interjacent sequences between the combined genetic
elements, the assay shows sufficient sensitivity and linearity. In addition, the method has
demonstrated to be transferable to other laboratories and to be robust enough for the use
with different real-time PCR machines and different PCR mastermixes.
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4. Literature
1. ISO/AOAC/IUPAC harmonized protocol in; Horwitz, W. (1995) Protocol for the design,
conduct and interpretation of method performance studies. Pure and Appl. Chem. 67:
331-343.
2. DIN EN ISO 21571:2005-05; Foodstuffs – Methods of analysis fort he detection of
genetically modified organisms and derived products – Nucleic acid extraction
3. Bruderer S, Leitner KE (2003). Genetically Modified (GM) Crops: molecular and
regulatory details. Version 2 (30/06/2003). BATS, Centre for Biosafety Assessment,
Technology and Sustainability. http://www.bats.ch/gmo-watch/GVO-report140703.pdf
4. Laube I, Hird H, Brodmann P, Ullmann S, Schöne-Michling M, Chisholm J, Broll H (2008)
Development of primer and probe sets for the detection of plant species in honey. Food
Chemistry 118, 979-986
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