[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
[SANET-MG] Bt10 detection flawed and biased
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
The validation report  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
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
The GeneScan website has a link to a page on Syngenta’s website, which
advertises the “European Union Bt10 Detection Method”  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 , “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 , “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  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  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
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
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 .
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
• Full genetic map and base sequence of the Bt10 insert(s) including the
ampicillin resistance marker gene and the host genome sequences flanking
• 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
1. “EU detection method for Bt10 maize validated” European Commission
Health & Consumer Protection Directorate-General E-News 25-05-2005
2. 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.
3. European Union Bt10 detection method. Syngenta
4. PCR assay for detection of maize transgenic event Bt10. European
Commission. Community Reference Laboratory for GM food and Feed.
5. 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.
6. 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
To unsubscribe from SANET-MG:
1- Visit http://lists.sare.org/archives/sanet-mg.html to unsubscribe or;
2- Send a message to <firstname.lastname@example.org> from the address subscribed to the list. Type "unsubscribe sanet-mg" in the body of the message.
Visit the SANET-MG archives at: http://lists.sare.org/archives/sanet-mg.html
For more information on grants and other resources available through the SARE program, please visit http://www.sare.org.