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DOI 10.1007/s00217-006-0467-x
ORIGINAL PAPER
Received: 19 June 2006 / Revised: 14 August 2006 / Accepted: 21 August 2006 / Published online: 7 October 2006
C Springer-Verlag 2006
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272 Eur Food Res Technol (2006) 224:271278
Kemingdao, have entered pre-production trials in 2001 DNA was extracted from ground material using a modified
[7, 8]. CTAB nucleic acid extraction method [13]. 1.5 mL CTAB ex-
Last year the internationally operating environmental traction buffer (20 g/L cetyl-trimethyl-ammonium bromide,
group Greenpeace investigated the current marketing of GM 1.4 mol/L NaCl, 0.1 mol/L TrisHCl, 0.02 mol/L Na2 EDTA,
rice varieties in China [9]. Several rice seed samples were pH 8.0) and 10 L Proteinase K solution (20 mg/mL) were
bought from local seed companies, at agriculture extension added to 200 mg of the milled rice sample. The samples
stations and from farmers in Central Chinas Hubei Province. were incubated at 60 C under constant agitation overnight
The collected samples were analysed by a private GMO- and then centrifuged for 10 min at 13,000 g. The super-
testing laboratory using DNA and protein based tests. In natant was transferred into a new vial, 750 L of chloro-
19 out of 25 samples the presence of transgenic rice could form was added, vigorously shaken and then centrifuged
be shown [10]. Several samples tested positive in the DNA at 13,000 g for 5 min. The upper phase was transferred
screening tests and were also positive in a Cry1A(c)-specific into a new vial, its volume was determined and mixed with
protein test, suggesting that these rice varieties express the two volumes of CTAB precipitation buffer (5 g/L cetyl-
Bt protein. trimethyl-ammonium bromide, 40 mmol/L NaCl). After in-
Analytical methods to detect GMOs and to determine cubation for 60 min at room temperature without agitation,
their content in food or feed are mainly DNA-based using the the samples were centrifuged for 15 min at 13,000 g, the
polymerase chain reaction (PCR). Due to its high specificity, supernatant was discarded and the pellet was resuspended
ease of use and applicability both as qualitative and as in 350 L of a 350 mmol/L NaCl solution. Chloroform
quantitative method, real-time PCR is the preferred approach (350 L) was added, the samples were mixed on a Vortex
to perform GMO-testing. Most real-time PCR detection and centrifuged for 10 min at 13,000 g. The upper phase
methods are based on the hydrolysis of fluorescence-labelled was combined with 0.6 volumes of isopropanol for nucleic
oligonucleotide probes (TaqManTM ) during each cycle of the acid precipitation and after 20 min incubation at room tem-
amplification process [11]. This method is also preferred by perature the samples were centrifuged 10 min at 13,000 g.
the EU in order to meet the recommended way to determine The supernatant was discarded, the pellet was washed with
the GMO content on the basis of haploid genome copies 500 L ethanol solution (70% EtOH) and resolved in 200 L
[12]. In order to develop detection methods for specific GM TE buffer (1 mmol/L TrisHCl, 0.1 mmol/L Na2 EDTA,
crops, positive test material must be available. However, it pH 8.0).
is extremely difficult to obtain authentic reference material The extracted DNA was quantified in a fluorometric as-
for those GM varieties which are not developed and planted say using the PicoGreen dsDNA binding dye (Invitrogen)
in the EU or for which no official EU authorisation is according to the manufacturers instructions. Measurements
applied. were conducted in an ABI PRISM 7900 (Applied Biosys-
In an attempt to develop a detection method for GM rice tems) at 525 nm. A 100 bp molecular size DNA marker with
varieties which are in the process of commercialization in a concentration of 0.1 g/L DNA (Fermentas) was used
China but currently not approved in the EU, we used two for calibration.
rice seed samples of the Greenpeace survey as reference For specificity tests, DNA was extracted from two com-
material. Based on the assumption that the material contains mercially available conventional rice brands bought in Ger-
GM Shanyou 63 rice, parts of the transgenic sequences many (Wurzener Parboiled Reis, Lot L 60531E, Wurzen,
were amplified to develop a construct-specific real-time PCR Germany and Gut & Gunstig Spitzen-Langkorn-Reis, Lot
assay. We here describe a method for detection of Bt rice 07\02\2008, Hamburg, Germany), from GM rice line LL-
varieties which could be also used for quantification of the RICE62, GM soya line GTS40-3-2 and GM maize lines T25,
content if present in imported food products. Bt11, Bt10, Bt176, MON810, MON863, NK603, TC1507,
GA21 and CBH351.
For sensitivity tests, a serial dilution of DNA extracted
Material and methods from samples FR0502519 and FR0502520 was prepared by
stepwise fourfold dilution with 0.1 TE buffer. Another
Sample material and DNA extraction serial dilution also used to test for the sensitivity was pre-
pared in the same way with DNA extracted from sample
Two GM rice samples, FR0502519 and FR0502520, were FR0502519 and a mixture of genomic DNA from conven-
kindly provided by Greenpeace International who collected tional rice (Wurzener Parboiled Reis, 10 g/mL) and maize
the packaged rice seeds in an agriculture extension station in (10 g/mL). The mass fraction mixtures with different GMO
Wangjiaqiao Town, Songzi City in Hubei Province, China, contents were prepared using ground conventional rice and
in spring 2005 [10]. The samples were labelled as Anti-pest GM rice sample FR0502519. A mixture of 9.5 g conven-
Shanyou 63 and as Anti-pest Jinyou 63. tional rice and 0.5 g of rice sample FR0502519 resulting
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Eur Food Res Technol (2006) 224:271278 273
in a 5% (w/w) sample was used a starting material. 2 g of quencing was done by a primer walking strategy to generate
this 5% (w/w) mixture was added to 8 g of conventional sequences in overlapping regions of the different construct
rice giving a 1% (w/w) sample. A subsequent 0.5% (w/w) elements. Nucleic acid sequence data were first compared
mixture was prepared using 5 g of the 1% (w/w) sample and with the Sequence Navigator software (Applied Biosystems)
5 g of conventional rice, and a 0.1% (w/w) level was pre- and then analysed by searches in the GenBank sequence
pared using 8 g conventional rice and 2 g of the 0.5% (w/w) database using the computer algorithm BLAST 2 [19].
sample. For subsequent quantitative real-time PCR analyses
DNA was extracted from the 5, 0.5 and 0.1% mass fraction Real-time PCR
mixtures.
Real-time PCR was performed in an ABI PRISM 7900 in-
PCR and DNA sequencing strument (Applied Biosystems). All reactions were run as
duplicates in 96-well plates. The 25 L reaction mixtures
PCR was performed in a volume of 25 L containing 2.5 L contained 12.5 L universal master mix (Applied Biosys-
10 PCR buffer with 15 mmol/L MgCl2 , 0.5 mol/L of tems), the indicated concentrations of primers and probe
each primer, 0.625 U Taq polymerase (HotStar, Qiagen) and (Table 1) and 5 L of template DNA. The reaction conditions
1 L of template DNA corresponding to 25 ng DNA. For were as follows: Initiation step for 10 min at 95 C followed
thermal cycling, an initial denaturation step for 15 min at by 45 cycles of 20 s at 95 C and 1 min at 60 C. Primers
95 C was followed by 45 cycles of 30 s at 94 C, 30 s at T51F and T51R1 and probe T51p (Table 1) were designed
60 C and 60 s at 72 C with a final elongation step of 7 min with the Primer Express 2.0 software (Applied Biosystems).
at 72 C. In conventional PCR and real-time PCR targeting Primer and probe sequences for detection of taxon-specific
the CaMV 35S promoter and the nos terminator sequences rice reference genes were taken from published real-time
protocols described elsewhere were used [1418]. PCR assays [20, 21].
For sequence determination of the transgenic construct
DNA extracted from samples FR0502519 and FR0502520
was used in PCR experiments with primers RiceActin1f (5 - Results and discussion
ccc tct cct ctt tct ttc tcc g-3 ; personal communication N.
Hess) in combination with primer NOS180R (5 -TTg TTT Sequence analysis of GM rice samples
TCT ATC gCg TAT TAA ATg T-3 ; personal communication
R. Reiting) at reaction conditions as described. Additional Two rice seed samples taken at local Chinese wholesalers by
PCR products were generated with oligonucleotides NOS-1 Greenpeace in the year 2005 were used for DNA sequence
(5 -gAA TCC TgT TgC Cgg TCT Tg-3 ), NOS-3 (5 -TTA analysis. These samples, FR0502519 and FR0502520, were
TCC TAg TTT gCg CgC TA-3 ) and CryIA(b)F (5 -ACC initially investigated with GMO screening tests. Both sam-
ATC AAC AgC CgC TAC AAC gAC C-3 ) using reaction ples showed positive reactions in a CryIA(c) immuno-based
conditions described elsewhere [14, 17, 18]. Bt-protein test and in a DNA-based test for the nopaline syn-
PCR products were purified with a QIAquick PCR purifi- thase (nos) transcription terminator sequence, whereas only
cation kit (Qiagen) and directly sequenced with the BigDye one sample (FR0502520) was positive for the CaMV 35S
Terminator V 1.1 cycle sequencing kit (Applied Biosystems) promoter sequence in a 35S DNA-test [10]. Re-investigation
in an ABI PRISM 310 instrument (Applied Biosystems). Se- of these results with a 35S promoter-specific real-time PCR
Table 1 Description of the different real-time PCR systems used in the study. The length of the generated PCR product is given in brackets
Final concentration
Method Name Oligonucleotide sequence (5 3 ) in PCR (nmol/L) Reference
Bt rice construct (83 bp) T51F gAC TgC Tgg AgT gAT TAT CgA CAg A 300 This work
T51R AgC TCg gTA CCT CgA CTT ATT CAg 300
T51p FAM-TCg AgT TCA TTC CAg TTA CTg CAA CAC 100
TCg Ag-TAMRA
gos9 rice reference gene (68 bp) org1 TTA gCC TCC CgC TgC AgA 300 [21]
org2 AgA gTC CAC AAg TgC TCC Cg 300
orgp FAM-Cgg CAg TgT ggT Tgg TTT CTT Cgg-Dabcyl 100
sps rice reference gene (81 bp) SPSF TTg CgC CTg AAC ggA TAT 750 [20]
SPSR Cgg TTg ATC TTT TCg ggA Tg 750
SPSP FAM-gAC gCA Cgg ACg gCT Cgg A-Dabcyl 300
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274 Eur Food Res Technol (2006) 224:271278
Fig. 1 Diagram of the construct pFHBT1 inserted in GM Shanyou cated by the arrows. The location of sequences taken for the alignments
63 according to Tu et al. [7]. The positions of the transgenic elements, shown in Fig. 2 and Fig. 3 are indicated by the striped bars. The position
their sizes and the restriction sites are given. Primers used in the present of the construct-specific Bt rice detection method with its primers and
study to generate PCR products suitable for DNA sequencing are indi- probe are also shown below the right striped bar
test showed that only a weak 35S reaction with a Ct-value in sample FR0502520, since the construct in the suspected
of 35 is detectable with DNA from FR0502520 when GM Shanyou 63 line should not contain the CaMV 35S
compared to the Ct-value of 24 obtained in the nos- promoter and the removal of the 35S promoter-driven hph
specific real-time PCR using the same amount of sample marker gene by segregation has been reported for the parental
DNA. We assume that traces of another GMO is present CMS restorer line Minghui 63 [22].
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Eur Food Res Technol (2006) 224:271278 275
Several different primer combinations targeting the pre- not present in the deduced protein sequence of the identified
sumed transgenic construct inserted in GM Shanyou 63 [7] reading frame [23].
were used to generate PCR products suitable for direct DNA To further analyse the transgenic sequences in the rice
sequencing (Fig. 1). The nucleotide (nt) sequences of these sample materials under investigation, it was tested whether
products were analysed with the BLAST similarity search the nos terminator is also located behind the Bt cryIA(b) and
engine of the National Center for Biotechnology Informa- cryIA(c) fusion gene as described for the pFHBT1 construct
tion (NCBI). The two regions selected for alignments of the [7, 22]. Using DNA from FR0502519 and FR0502520 as
transgenic sequences with identical GenBank sequences are template, distinct PCR products were amplified with primers
shown (Figs. 2 and 3). The 5 sequence part of the ampli- spanning the region of the 3 part of the cryIA(c) gene and
fied fragment shows complete identity to the rice Act1 intron the nos terminator (see Fig. 1). Sequencing results shows
contained in a plant transformation vector (pPLEX-5013) that in this region no differences are present in the two in-
over a stretch of 404 nucleotides (Fig. 2). This sequence is vestigated rice samples (Fig. 3). The first 393 nucleotides
followed by a 38 nt long spacer with fragments of a multiple of the sequence completely match to a GenBank database
cloning site until a potential ATG start codon of the Bt toxin entry coding for a synthetic cryIA(c) gene. At the end of
gene encoding reading frame is found. The next 347 nt long the cryIA(c) sequence homology a 26 nt long spacer with
stretch shows identity to a synthetic CryIA(c) gene (accession no similarity to known sequences is located, followed by a
number Y09787). The sequence of the PCR products gen- 169 nt long sequence identical to the nos terminator derived
erated with DNA from FR0502519 and FR0502520 showed from Agrobacterium tumefaciens.
no differences in this region (data not shown), indicating
that either both samples represent the same GM rice event, Construct-specific detection of Bt rice varieties
or that both samples derive from transformation events with
the same construct. The identified transgenic construct pre- To detect all Bt rice lines transformed with plasmid construct
sumably corresponds to plasmid pFHBT1 which was used pFHBT1 which are possibly marketed in China we decided
for production of the GM Shanyou 63 line [7, 22]. Partic- to develop a construct-specific detection method instead of
ularly a short amino-terminal peptide sequence (P-N-I-N-E- an event-specific assay. Based on the sequence data deter-
C-I) deleted in the hybrid cryIA(c) gene of pFHBT1 is also mined from the two rice samples the transition between the
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276 Eur Food Res Technol (2006) 224:271278
Table 2 Sensitivity and detection range of the construct-specific Bt ing genome copy number was calculated on basis of the mass of the
rice real-time PCR system. Serial dilutions of DNAs from rice samples rice genome of 0,47 pg [25]
FR0502519 and FR0502520 were tested in duplicates, the correspond-
Sample DNA concentration
FR0502519 Amount of DNA (pg) 12,800 3,200 800 200 50 12.5 3.1 0.8
Haploid genome copy number 27,198 6,800 1,700 425 106 27 7 2
Ct-value (mean) 26.6 28.4 31.1 33.3 35.6 38.8 40.2 n.d.
FR0502520 Amount of DNA (pg) 13,200 3,300 825 206 52 13 3.2 0.8
Haploid genome copy number 28,100 7,025 1756 439 110 27 7 2
Ct-value (mean) 26.9 28.8 31.3 33.3 35.8 38.0 39.2 42.6 / n.d.
synthetic cryIA(c) and the nos terminator was used to design negative PCR results were obtained (Table 2). Based on the
a PCR detection system (Fig. 3). To ensure that only this assumption that one haploid rice genome copy has a molecu-
83 bp long fragment of the construct will be amplified, one lar mass of 0.47 pg [24], the detection limit of the construct-
of the primers is located directly in the spacer sequence. The specific Bt rice detection system is seven copies, also in pres-
specificity of this PCR system was then tested with approx- ence of conventional rice and maize DNA in the test reaction
imately 10100 ng DNA extracted from several GM plant (data not shown). Using the mean Ct-values of each dilution
reference materials (LL62 rice, GTS40-3-2 soya, T25, Bt11, step the corresponding regression curve was calculated (Fig.
Bt10, Bt176, MON810, MON863, NK603, TC1507, GA21 4). The Ct-values measured with the construct-specific Bt
and CBH351 maize) and from conventional rice varieties. rice system were also compared with those obtained in the
The amplifiability of DNAs was previously checked using nos real-time PCR. A dilution series with five steps of four-
taxon-specifc reference gene detection systems for soya and fold DNA dilutions of samples FR0502519 and FR0502520
maize [16]. None of the conventional and of the GM lines was tested. The Bt rice system showed Ct-values of 29.5
including the Bt maize varieties reacted positive with the to 37.8 for FR0502519 and of 29.1 to 39.1 for FR0502520,
construct-specific Bt rice PCR system (data not shown). respectively. Using the nos-specific system, Ct-values ob-
tained with FR0502519 DNA ranged from 29.9 to 37.5 and
Sensitivity and detection limit from 29.5 to 41.3 with FR0502520 DNA, respectively. The
fluctuation of the Ct-values observed with low DNA copy
The detection limit of the Bt rice real-time PCR assay numbers at the end of the dilution series can be attributed to
was then assessed by means of a DNA dilution series until the statistical distribution of DNA target molecules in these
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Eur Food Res Technol (2006) 224:271278 277
Table 3 Reproducibility of the PCR assay tested in 4 parallel DNA earlier than the sps system when using the same amount of
extractions from materials containing mixtures with different Bt rice template DNA. A DNA dilution series of sample FR0502520
mass fractions. The gos9 target sequence was used to determine the
amount of rice reference gene copies, the cry1A(c)/nos target sequence tested in the gos9 real-time PCR system yielded Ct-values
represents the construct-specific Bt rice detection system described in ranging from Ct 28.9 to Ct 36.8, whereas the same DNA
the present study. analysed with the sps real-time PCR system showed Ct-
Ct-values measured with Bt rice values ranging from Ct 33.5 to Ct 43.9. Therefore, it was
content (w/w) of decided to select gos9 as rice reference gene target and to use
DNA extraction Gene target 5% 0.5% 0.1% this detection system for quantitative Bt rice real-time PCR
analysis.
1 gos9 24.5 24.8 24.8
The sensitivity of this quantitative Bt rice assay was then
cry1A(c)/nos 29.8 33.8 36.2
2 gos9 23.8 24.0 24.9 tested with different mass fraction mixtures of conventional
cry1A(c)/nos 30.2 33.7 36.5 rice and rice sample FR0502519. Four DNA extractions were
3 gos9 23.8 24.4 23.8 done in parallel from the mixtures containing 5, 0.5 and 0.1%
cry1A(c)/nos 29.7 34.2 37.4 Bt rice FR0502519 and each DNA sample was analysed
4 gos9 23.7 24.1 25.1 in duplicate (Table 3). The results show that low GM rice
cry1A(c)/nos 30.2 34.8 36.3 concentrations of 0.1% could be reproducibly detected
and that the method should allow an estimate of the relative
GM rice content.
samples. The results indicate that the nos- and the Bt-specific
real-time PCR system yield comparable Ct-values with equal Detection of unapproved GM rice varieties
DNA samples, suggesting that FR0502519 and FR0502520
are not mixed with other GM rice seeds and that nos and the Cases of marketing and the use of GM rice varieties apart
Bt construct are presumably present in the same ratio. from deliberate field release trials is increasing in China and
In order to establish a detection assay applicable for rela- the effectiveness of the Chinese legislative and administrative
tive quantification, the construct-specific Bt rice PCR system frame work for surveillance to prevent uncontrolled cultiva-
was combined with a target taxon-specific real-time PCR tion of genetically modified crops is publicly debated [3, 9,
method. Recently, the validation of two different rice ref- 25]. Although the identity of the material used in our study
erence gene-specific real-time PCR methods has been de- is not exactly known, the analysis of the transgenic DNA
scribed [20, 21]. The method for detection of the sps gene sequences suggests that it contains the genetically modified
which codes for the sucrose phosphate synthase amplifies a rice line TT51-1, derived from line Minghui 63, and the
81 bp long fragment of the rice genome (Table 1). Speci- resulting hybrid rice line GM Shanyou 63 [7]. The general
ficity tests with 13 different rice varieties showed that the analytical strategy of the official European GMO control
sps gene contains no allelic variations in the amplified re- laboratories is first to perform DNA screening tests targeting
gion [20]. The detection limit of the sps PCR system was elements which are widely used in GM plants and then in
reported to be 10 DNA copies. The rice reference gene PCR the next analysis step to identify the GMO event. With the
system reported by Hernandez et al. [21] amplifies a 68 bp commonly used CaMV 35S promoter and the nos terminator
long fragment of the root-specific gos9 rice gene with a de- DNA screening methods several GM rice varieties, including
tection limit of three copies. We tested both methods at the GM Shanyou 63, should be detectable (Table 4). Because
cycling conditions of the construct-specific Bt rice method. the construct-specific Bt rice method described in the present
At 60 C as annealing/elongation temperature with a two- study targets DNA sequences that should be only present in
step cycling protocol, the gos9 system was found to be more GM-derived rice, it may help to confirm positive 35S/nos
sensitive when compared to the sps system. The gos9 sys- DNA screening results obtained in routine GMO analyses of
tem showed amplification curves appearing at about five Ct food products that otherwise could not be attributed to any
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278 Eur Food Res Technol (2006) 224:271278
GM plant. The comparison of the two different rice reference 13. ISO 21571:2005 Foodstuffs. Methods of analysis for the detec-
gene detection methods shows, that the gos9 real-time PCR tion of genetically modified organisms and derived products nu-
cleic acid extraction. International Standardization Organisation,
system is compatible to a two-step PCR protocol and the cy- Geneva, Switzerland
cling conditions used for the Bt rice real-time PCR method. 14. Official Collection of Test Methods (2001) Screening for detection
We therefore suggest that this complete assay is applicable of genetically modified DNA sequences in foodstuffs by detec-
also to quantitatively detect unauthorised Bt rice varieties in tion of DNA sequences which are frequently present in genetically
modified organisms German Federal Foodstuffs Act Food Anal-
EU food imports. ysis, Article 35, L 00.00-31, Beuth-Verlag, Berlin
15. ISO 21569:2005. Foodstuffs methods of analysis for the detection
Acknowledgements We thank Dr. Janet Cotter (University of Ex- of genetically modified organisms and derived products quali-
eter, Exeter, UK) for providing us with the rice sample materials. tative nucleic acid based methods. International Standardization
We also thank Dr. Joachim Bendiek (Bundesamt fur Verbraucher- Organisation, Geneva, Switzerland
schutz und Lebensmittelsicherheit, Berlin) for critical reading of the 16. ISO 21570:2006. Foodstuffs methods of analysis for the detection
manuscript. of genetically modified organisms and derived products quanti-
tative nucleic acid based methods. International Standardization
Organisation, Geneva, Switzerland
17. Pietsch K, Waiblinger HU, Brodmann P, Wurz A (1997) Dtsch
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