ISIS Press Release 18/05/05
Bt10 Detection Method Unacceptable
The detection method for Syngenta’s illegal GM maize is flawed;
there must now be a full disclosure of information and access to reference
material for retrospective risk assessment and risk management.
Dr. Mae-Wan Ho and
Prof. Joe Cummins
Concerted move to reassure the European public
Swiss biotech firm Syngenta had accidentally sold illegal GM maize Bt10
in the US for the past four years, resulting in about 133 million kilograms of
the maize making its way into food and feed.
The news broke on 22 March 2005 in the science journal Nature
("Syngenta’s GM maize scandals",
SiS 26),
although Syngenta had entered into talks with the US government since December
2004.
Under pressure from public protests across the world, the US government
fined Syngenta a derisory US$375 000 (euro 270 000) for the mishap. And on 18
April, the European Commission imposed an emergency measure to ban certain GM
maize imports from the US unless they are accompanied by an original analytical
report issued by an accredited laboratory demonstrating that the product does
not contain Bt10 ("Europe acts swiftly to keep out unapproved GM maize",
SiS26).
Scarcely a week later, the EU authorities announced that Syngenta had
presented a detection test for Bt10, which was already validated by the EU
authorities.
The validation report [2] from the Joint Research Centre, also
Europe’s Community Reference Laboratory (CRL) for GM Food and Feed, said
it carried out an in-house validation of the event-specific detection method
"proposed by GeneScan on Bt10 maize developed by Syngenta Crop Protection AG."
Syngenta provided the DNA samples (genomic DNA extracted from the Bt10
maize line and from a control maize line), and GeneScan provided the
event-specific detection method based on a qualitative polymerase chain
reaction (PCR) assay.
Monopoly on detection method declared
So who, or what is GeneScan? GeneScan advertises itself on its
website as "the world market leader in the field of molecular biological
testing for Genetically Modified Organisms (GMOs) in food, feed and
agricultural raw materials."
The GeneScan website has a link to a page on Syngenta’s website,
which advertises the "European Union Bt10 Detection Method" [3] as a "validated
detection methodology that has been thoroughly tested for accuracy, reliability
and sensitivity" using authentic samples to ensure actual targeted material is
detected reliably when present. The method is designed, it says, to exclude
"false positives" in the hands of "highly qualified scientific personnel with
specific experience with the protocol", working under "exemplary laboratory
practice and standard operation procedures (SOPS) from an …accredited
lab", with "provisions for retesting false positives".
The same Syngenta page advises us that GeneScan is "the only private
service laboratory that fulfils the elements listed above for Bt10 testing",
and the fact that the EU Joint Research Centre has certified the GeneScan
method on April 22, 2005 as "the only EU official method for Bt10 detection."
Following that, yet again, the admonition to guard against "false positives" is
repeated.
In contrast, there’s not a word said about false negatives, which
as every molecular geneticist knows, is also a problem with the PCR detection
method, particularly if the GM insert is unstable, and prone to deletions and
rearrangements, as revealed in recent analyses by European government
laboratories ("Transgenic lines proven unstable",
SiS20;
"Unstable transgenic lines illegal",
SiS21).
This three-way mutual reinforcement between Europe’s Joint Research
Centre (the European Commission’s official laboratory), Syngenta and
GeneScan seems just a bit too cosy to be reassuring. What’s more, they
have jointly declared a monopoly on the detection method, ruling out all others
that could give "false positives". It is a case of the poacher turned
gamekeeper with the help of the governor.
The validation report issued by the Joint Research Centre (JRC) goes on
to state [2], "The results of the JRC validation demonstrated that the method
reliably detects an amplification product specific for Bt10 maize, and
therefore allows discriminating event Bt10 from other GM-events in maize lines.
The sensitivity of the method is below 0.1%….
"The method is therefore considered by the CRL as fit for the purpose
of Bt10 detection and it is the only accepted to certify the presence of
Bt10 in maize commodities in accordance with the Commission Decision
1005/317/EC). (emphasis added)
When is a positive false?
In fact, the method amplifies and detects a small 130base pair fragment
of Bt10 DNA, said to be specific for Bt10. It is not stated which gene fragment
from Bt10 is being amplified. A strict protocol is laid out in detail. The Bt10
and wild type DNA supplied by Syngenta were analysed along with other reference
and non-reference material contained in the JRC’s Community Reference
Laboratory.
The 130 bp band was indeed specifically amplified only in Bt10. But
unfortunately, bigger bands were amplified and detected in other GM maize
lines, and even in the wild-type maize DNA supplied by Syngenta. Strangely
enough, these higher molecular weight bands were absent from the Bt10 DNA from
Syngenta.
The origins of the "unspecific amplicons" (amplified DNA) were not
investigated further, but effectively dismissed with the remark, "This suggests
that the method can be further optimised." Consequently, only the 130bp
amplicon is regarded as a definite positive.
The conclusion of the validation report states that the method is "fit
for its intended purpose", with the qualification [3], "However, at this stage
of testing, the method produces a higher molecular-weight multi-band pattern in
GM and non-GM maize which requires additional efforts in its optimisation."
Still further qualifications are contained in a later report [4] on the
detection method: "The analyst shall be aware that other validation experiments
indicated that the method might perform less reliably at annealing temperatures
higher than specified in the protocol. Moreover, in some incidents unspecific
amplification was observed with PCR profiles that used high numbers of cycles
than specified in the protocol. Time constraints did not permit to rectify
these concerns…"
As mentioned earlier, fragmentation or rearrangements of the GM insert
can change the size of the amplicon, or otherwise fail to give the specific
amplicon. Consequently, unless fragmentation or rearrangement of the Bt10 GM
insert can be ruled out, it is not legitimate to conclude that amplicons of
other sizes are "false positives".
Further data, further confusion
Syngenta’s reports sent to the US Environment Protection Agency
earlier this year have been leaked to ISIS.
The first report dated 28 January 2005 [5] is intended to present the
DNA sequence of Bt10 compared with Bt11, the GM maize line that Bt10 had
contaminated by accident. The Bt10 insert was mapped to chromosome 1 of the
maize genome, while Bt11 insert had been mapped to chromosome 8. This alone
will indicate that Bt10 is completely different from Bt11. In addition, there
were three nucleotide changes in Bt10 compared with Bt11: two in an unspecified
sequence contained within the Bt10 insert (unspecified sequence 1 in Figure 1
below), and one located in the nos terminator associated with the
crylAb gene. No nucleotide changes were identified in any of the coding
sequences and promoters within the Bt10 insert.
However, the map of the Bt10 insert presented can only be partial, as
it did not include the ampicillin antibiotic resistance marker gene, unless
that marker gene has inserted elsewhere in the genome. The map presented also
contained at least three unspecified, unknown sequences (Fig. 1).
Unspecified sequence 1 (>1000 bp)-p35S
(516pb)-IVS6 maize adh1S (477bp)-crylAb(syn)
(1848bp)-tnos (267bp)-Unspecified sequence 2
(~400bp)-p35S(422bp)-IVS2 maize adh1S (180bp)-pat
(522bp)-tnos (259 bp)-unspecified sequence 3 (~160bp)
Figure 1. Map of Bt11 from Syngenta’s report to US EPA
The second report from Syngenta to the EPA is of a study comparing the
transgenic proteins expressed in Bt10 compared with those in Bt11 [6]. The
proteins were extracted from leaves of the plants, and subjected to western
blot analyses, a technique dependent on staining the protein bands with
specific antibodies after separating them by migration in an electric field
through a gel matrix.
This report claims that the analyses "revealed similar dominant
immunoreactive bands" in both Bt11 and Bt10 corresponding to the predicted
Cry1Ab protein (for insect resistance) and phosphinothricin acetyltransferase
(PAT) (for tolerance to the herbicide glufosinate ammonium) of about 69 000 and
22 000 daltons respectively.
However, the photographs of the western blots contained in the report
tell a different story. Bt11 showed a series of bands at 46 000, 63 000 and 52
000 daltons (in order of strength of staining) besides the dominant 63 000
daltons band, whereas Bt 10 only had the 63 000 daltons fragment besides the
main predicted band. The PAT protein bands in Bt10 and Bt 11 were also
different from each other and from the purified standard, with many high
molecular weight bands reacting to the antibody.
Neither report contains information on the breeding history of the GM
maize lines analysed, such as the number of generations since the
transformation event; nor data from appropriate reference material. These are
sure signs of sloppy science.
Full disclosure of molecular data and access to reference material
required
The detection method for Bt10 is flawed by the admission of the European
authorities. The identity of the 130 bp amplicon, supposed to be specific for
Bt10, is not made explicit. The molecular data supplied to the US EPA are
incomplete. It is impossible to judge if the detection method is adequate in
the absence of full molecular data including those from reference material
proving that Bt10 had remained genetically stable since it was first
unintentionally released.
Bt11 had already been exposed to be unstable, and to be contaminated
with another Syngenta maize Bt176, implicated in the death of dairy cows in
Hesse Germany ("Cows ate GM maize and died",
SiS 21).
Syngenta has admitted that Bt10, as distinct from Bt11, contains an
ampicillin resistance marker gene, which, according to an Opinion issued by the
Scientific Panel on Genetically Modified Organisms of the European Food Safety
Authority in 2004,
"should not be present in GM plants to be placed on the market". No
official information has been forthcoming regarding the ampicillin resistance
marker gene in Bt10, nor any attempt to ascertain whether the marker gene has
contaminated other maize varieties, GM or otherwise.
As Bt10 has already entered the market and the human food chain,
it must go through retrospectively the risk assessment process that would have
been applied to a GM product approved for market. This is also essential for
effective post-release risk management.
At the very least, Syngenta must be required to provide the
following:
- Reference plant material from successive generations of the Bt10
transformation event plus the non-GM maize variety from which Bt10 was
derived
- Full genetic map and base sequence of the Bt10 insert(s) including
the ampicillin resistance marker gene and the host genome sequences flanking
the insert(s)
- Genome location of the Bt10 insert(s)
- Profiles of expressed RNAs and proteins in the Bt10 reference
material, compared to those in Bt11 and the non-GM variety or varieties from
which the GM maize lines were derived
- Molecular genetic data of at least five generations after the Bt10
transformation event, to document genetic stability
- Any other information available on Bt 10
Furthermore, regulatory authorities on both sides of the Atlantic must
make public all information on Bt10 that they have received from Syngenta or
other sources.
Please circulate this report widely and send it to your elected
representatives.
- "EU detection method for Bt10 maize validated" European Commission
Health & Consumer Protection Directorate-General E-News 25-05-2005
http://europa.eu.int/comm/dgs/health_consumer/dyna/enews/enews.cfm?al_id=18
- Mazzara M, Maretti M, Foti N, Price S, Paoletti C, Savini C and Van
den Eede G. Joint Research Centre – European Commission. Report on the
in-house validation of a detection method for event Bt 10 maize using a
qualitative PCR assay.
http://gmo-crl.jrc.it/detectionmethods/Bt10%20validation%20report.pdf
- European Union Bt10 detection method. Syngenta
http://www.syngenta.com/en/downloads/050427_Bt10_EU_Method.pdf
- PCR assay for detection of maize transgenic event Bt10. European
Commission. Community Reference Laboratory for GM food and Feed.
http://gmo-crl.jrc.it/detectionmethods/Bt10%20Detection%20Protocol.pdf
- Rabe,s,Mumm,R.Shi,L. and Stein,J. Sequencing of the Bt10 insert and
comparison with the previously reported Bt11 sequence Syngenta
Biotechnology,Inc. Report : SSB-104-05, January 28,2005.
- Graser G. Western blot analysis of CrylAb and PAT proteins expressed
in field corn. Report No. SSB-112-05. Syngenta report to US EPA, 11 February
2005.
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