The development of a new screening method depends on several prerequisites: information on the elements inserted in a GM event, their copy number and the nucleotide sequence of the inserted element. Information on the elements of the transgenic construct inserted in a GMO can be obtained from publicly available dossiers submitted by the applicant for authorisation or patent databases. This information is usually available after the authorisation is granted or after the competent authorities have given a positive advice. Important information sources are the GMO crop database of the Centre for Environmental Risk Assessment (CERA) (http://www.cera-gmc.org/?action=gm\_crop\_database) and the GMO database on authorisations and approval of GMO in the EU (http://www.gmocompass.org/eng/gmo/db/). The nucleotide sequences are available in public databases such as the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), patent databases and scientific publications. One must however take care when using the information present in these databases as for example Single Nucleotide Polymorphisms (SNP) may exist in the sequence of the elements inserted in different GM events (Morisset et al., 2009). Therefore, the information in the public databases is not always completely reliable and more than one source should be consulted. Additionally, variations in the sequences used to design taxon-specific assays exists as for instance SNP can occur between the varieties of one plant species (Broothaerts et al., 2008; Papazova et al., 2010). The difficulty here is that information on the nucleotide sequence in different plant varieties is not available. This problem can be partially solved by designing the SYBR®Green primers on basis of existing TaqMan® taxon-specific assays for which experimental tests have been performed. Presence of SNP in the primer annealing sites can lead to a false negative result and to the conclusion that an event containing this target is not present when the assay is applied to an unknown sample (Broothaerts et al., 2008; Papazova

Upon selection of the suitable sequence different primer pairs are designed by using appropriate bioinformatic tools. One of the most widely used programs is Primer3 (Rozen & Scaletzky, 2001). These primer pairs are further assessed *in silico* for their specificity. This can be done by means of bioinformatic tools such as the primer search module in the EMBOSS bioinformatic platform, BLAST searches etc. For transgenic elements, this theoretical specificity test is performed using sequences from authorised GM events. If the primers target a reference taxon-specific sequence, it should be tested if they are specific for the target taxon and do not amplify closely related species. Here, the criteria for specificity

As the goal is to use all the methods simultaneously under uniform conditions, particular attention is paid on the amplicon size and the primer annealing temperature (Tm) when developing the primers. Amplicons with a size lower than 100 bp are preferred although the size for real-time PCR amplicons can be as large as 250 bp. For qPCR detection smaller amplicons are favoured in order to avoid lack of amplification due to the possible fragmented status of the DNA in the sample (part 2). In addition, the melting temperature of the primers should be around 60°C according to the general requirements for qPCR primers (www.appliedbiosystems.com). The formation of primer dimers and hairpins should be checked and primer pairs showing this feature should be excluded for further analysis.

for reference assays of the event-specific quantification methods also apply (part 5).

necessary to test for by the enforcement laboratories should be targeted.

The GMO platform developed by the WIV-ISP-GMOlab allows detection of authorised GM events as well as UGM in a cost- and time effective manner. It consists of a preparative DNA extraction step and three consecutive qPCR steps. The CoSYPS system, including in-house developed SYBR®Green screening methods, forms an innovative tool in GMO detection allowing reducing the number of identifications to be carried out. The TaqMan® identification further allows a narrowing down of the GM events present to a specific GMO and quantification permits the determination of the GM content.

## **4. Development and validation of a qualitative qPCR method in view of its application for screening purposes in the WIV-ISP GMO detection platform**

## **4.1 Introduction**

As described previously, in order to face the rapidly increasing number of GMO in food and feed products, new methods facilitating an initial screening of analytical samples is needed. Therefore, one of the major objectives of the molecular platform at WIV-ISP is to develop qualitative screening methods targeting either new genetic elements commonly found in transgenic constructs or species frequently used in food and feed in view of rationalizing GMO detection.

The methods developed are singleplex qPCR, based on SYBR®Green chemistry. Additionally, the methods are designed to work under uniform conditions (primer concentrations, PCR program) in order to facilitate their simultaneous application in a 96 well plate format. These SYBR®Green methods were in-house validated in order to be applied under ISO 17025 accreditation. As there is no 'golden standard' for the validation of qualitative methods related to GMO detection, enforcement laboratories need to decide which parameters need to be evaluated in the validation. In addition, the laboratories have to set their own criteria based on the guidance document for quantitative qPCR methods.

Part 4.3 of this chapter focuses on the method validation criteria and proposes a pragmatic approach for the in-house validation of singleplex real-time PCR qualitative methods. This proposal is mainly based on the recently adopted Codex Alimentarius guidelines on performance criteria and validation of methods for GMO analysis (Codex, 2010), and on the minimum performance requirements for methods for GMO testing set forward by the ENGL (ENGL, 2008). During the in-house validation critical values are determined for the screening methods to be introduced in the Decision Support System currently used in the routine analyses, namely the CoSYPS (part 6).

#### **4.2 Development of SYBR® Green methods for screening purposes**

The first step of method development is to determine the screening qPCR target. Targets for screening can be any element present in the transgenic construct inserted in authorised or unauthorised GMO and taxon-specific sequences. Application of the screening approach requires development of many targets in order to cover the growing range of GM events. Selection of the methods to be developed is based on a number of priorities. Firstly, methods targeting the main commodity crops used in transformation events are of high importance. Secondly, priority is given to transgenic elements frequently occurring in EU authorised GM events in addition to targets that provide an extra discriminative power. Thirdly, other

The GMO platform developed by the WIV-ISP-GMOlab allows detection of authorised GM events as well as UGM in a cost- and time effective manner. It consists of a preparative DNA extraction step and three consecutive qPCR steps. The CoSYPS system, including in-house developed SYBR®Green screening methods, forms an innovative tool in GMO detection allowing reducing the number of identifications to be carried out. The TaqMan® identification further allows a narrowing down of the GM events present to a specific GMO

**4. Development and validation of a qualitative qPCR method in view of its application for screening purposes in the WIV-ISP GMO detection platform**

As described previously, in order to face the rapidly increasing number of GMO in food and feed products, new methods facilitating an initial screening of analytical samples is needed. Therefore, one of the major objectives of the molecular platform at WIV-ISP is to develop qualitative screening methods targeting either new genetic elements commonly found in transgenic constructs or species frequently used in food and feed in view of rationalizing

The methods developed are singleplex qPCR, based on SYBR®Green chemistry. Additionally, the methods are designed to work under uniform conditions (primer concentrations, PCR program) in order to facilitate their simultaneous application in a 96 well plate format. These SYBR®Green methods were in-house validated in order to be applied under ISO 17025 accreditation. As there is no 'golden standard' for the validation of qualitative methods related to GMO detection, enforcement laboratories need to decide which parameters need to be evaluated in the validation. In addition, the laboratories have to set their own criteria based on the guidance document for quantitative qPCR

Part 4.3 of this chapter focuses on the method validation criteria and proposes a pragmatic approach for the in-house validation of singleplex real-time PCR qualitative methods. This proposal is mainly based on the recently adopted Codex Alimentarius guidelines on performance criteria and validation of methods for GMO analysis (Codex, 2010), and on the minimum performance requirements for methods for GMO testing set forward by the ENGL (ENGL, 2008). During the in-house validation critical values are determined for the screening methods to be introduced in the Decision Support System currently used in the

**Green methods for screening purposes**

The first step of method development is to determine the screening qPCR target. Targets for screening can be any element present in the transgenic construct inserted in authorised or unauthorised GMO and taxon-specific sequences. Application of the screening approach requires development of many targets in order to cover the growing range of GM events. Selection of the methods to be developed is based on a number of priorities. Firstly, methods targeting the main commodity crops used in transformation events are of high importance. Secondly, priority is given to transgenic elements frequently occurring in EU authorised GM events in addition to targets that provide an extra discriminative power. Thirdly, other

and quantification permits the determination of the GM content.

**4.1 Introduction**

GMO detection.

methods.

routine analyses, namely the CoSYPS (part 6).

**4.2 Development of SYBR®**

important transgenic elements occurring in unauthorised GM events which might be necessary to test for by the enforcement laboratories should be targeted.

The development of a new screening method depends on several prerequisites: information on the elements inserted in a GM event, their copy number and the nucleotide sequence of the inserted element. Information on the elements of the transgenic construct inserted in a GMO can be obtained from publicly available dossiers submitted by the applicant for authorisation or patent databases. This information is usually available after the authorisation is granted or after the competent authorities have given a positive advice. Important information sources are the GMO crop database of the Centre for Environmental Risk Assessment (CERA) (http://www.cera-gmc.org/?action=gm\_crop\_database) and the GMO database on authorisations and approval of GMO in the EU (http://www.gmocompass.org/eng/gmo/db/). The nucleotide sequences are available in public databases such as the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), patent databases and scientific publications. One must however take care when using the information present in these databases as for example Single Nucleotide Polymorphisms (SNP) may exist in the sequence of the elements inserted in different GM events (Morisset et al., 2009). Therefore, the information in the public databases is not always completely reliable and more than one source should be consulted.

Additionally, variations in the sequences used to design taxon-specific assays exists as for instance SNP can occur between the varieties of one plant species (Broothaerts et al., 2008; Papazova et al., 2010). The difficulty here is that information on the nucleotide sequence in different plant varieties is not available. This problem can be partially solved by designing the SYBR®Green primers on basis of existing TaqMan® taxon-specific assays for which experimental tests have been performed. Presence of SNP in the primer annealing sites can lead to a false negative result and to the conclusion that an event containing this target is not present when the assay is applied to an unknown sample (Broothaerts et al., 2008; Papazova et al., 2010).

Upon selection of the suitable sequence different primer pairs are designed by using appropriate bioinformatic tools. One of the most widely used programs is Primer3 (Rozen & Scaletzky, 2001). These primer pairs are further assessed *in silico* for their specificity. This can be done by means of bioinformatic tools such as the primer search module in the EMBOSS bioinformatic platform, BLAST searches etc. For transgenic elements, this theoretical specificity test is performed using sequences from authorised GM events. If the primers target a reference taxon-specific sequence, it should be tested if they are specific for the target taxon and do not amplify closely related species. Here, the criteria for specificity for reference assays of the event-specific quantification methods also apply (part 5).

As the goal is to use all the methods simultaneously under uniform conditions, particular attention is paid on the amplicon size and the primer annealing temperature (Tm) when developing the primers. Amplicons with a size lower than 100 bp are preferred although the size for real-time PCR amplicons can be as large as 250 bp. For qPCR detection smaller amplicons are favoured in order to avoid lack of amplification due to the possible fragmented status of the DNA in the sample (part 2). In addition, the melting temperature of the primers should be around 60°C according to the general requirements for qPCR primers (www.appliedbiosystems.com). The formation of primer dimers and hairpins should be checked and primer pairs showing this feature should be excluded for further analysis.

Development of a Molecular Platform

samples to the existing dataset.

unknown sample.

**4.4 Conclusion**

AFNOR XP V 03-020-2).

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 385

confidence interval can be updated regularly by adding data from analysis of routine

Using the data from the *in silico* and experimental specificity tests, mostly only one primer pair is selected for determination of the method sensitivity (LOD and LOQ) and repeatability. To assess the **sensitivity** of the developed method, a GM event containing the target is used (usually a CRM with a known GM%). It should however be noted that the GM-specific CRM are certified for the content of a specific GM event and not for the content of the screening target (promoter, coding sequence, terminator). This demonstrates that the preliminary information on the elements inserted in a GM event and their copy number is crucial in order to estimate the correct copy number of the target. For taxon-specific markers, this assessment can be done using a wild type (non-GM) material. The LOD and the LOQ are determined on basis of serial dilutions starting from at least 2000 target copies until the

The **LOD** is set up at the level where less than 5% false negatives are observed (Codex Alimentarius, 2009). As it is not feasible to perform the analysis on a large number of PCR replicates, six repeats are run per dilution point. If all six repeats are positive, this means that 95% of the time a positive sample will indeed be detected. Therefore the LOD of the screening method is set at the haploid genome copy level at which all six replicates provide

The **LOQ** is defined as the target copy number with a similar positive PCR result (expressed as Ct value) upon six-fold measurement of the target sequence in the same DNA sample with a minor standard deviation (SDCt<0,5) (AFNOR XP V 03-020-2). A screening target is in principle not quantified, but the LOQ can give an idea about the content of the target in an

Additionally, the **precision** (inter-run repeatability) of the method is determined. In practice this is done by calculating the relative repeatability standard deviation (RSDr%) on each of the dilutions used to determine the LOD and LOQ. Hereto, the experiment is performed under repeatability conditions (in a short period of time, on the same qPCR instrument by the same operator) in four independent runs. The RSDr% is calculated according to the

As, to date, no instructions on the development and validation of screening methods are available, the laboratories need to set up their own experimental plan and criteria. At the WIV-ISP-GMOlab, development and validation of SYBR®Green methods for screening purposes is done in a harmonized way to allow applying the methods in a single qPCR run. The parameters evaluated, the way to perform this assessment and the acceptance criteria are based on previously published documents (ENGL, 2008; Codex Alimentarius, 2009;

Upon evaluation of all the necessary parameters and their accordance with the set criteria, a validation dossier is established. The LOD, LOQ (expressed as a Ct value) and the Tm interval are introduced into the CoSYPS Decision Support System and serve as decision values to conclude if a sample is positive for the target or not (part 6). Subsequently the

theoretical zero copy numbers. Each of the dilutions is run in six replicates.

a specific positive signal (n = 6; 6/6 specific signals) (AFNOR XP V 03-020-2).

ISO 5725-2. The method is accepted as valid when the RSDr% is below 25%.

method is implemented in routine GMO detection under ISO 17025.

#### **4.3 Validation of a SYBR® Green screening method**

The in-house validation of a SYBR®Green screening method is based on the determination of several method characteristics that are required for the validation of event-specific quantitative methods (ENGL, 2008 - part 5), namely applicability, practicability, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ) and precision (RSDr%). The definitions of these parameters can be found in the glossary. The GMOlab has developed its own experimental set up in order to assess these parameters. Upon validation the results are evaluated and if they meet the acceptance criteria the method can be used under accreditation. Additionally, the critical values which are introduced in the CoSYPS (part 6) are determined during the in-house validation.

The method is **applicable** when it detects the target in the respective GMO for which it was designed. To test this aspect of a method a list of GM events containing the target (positive samples) and events not containing the target (negative samples) is made. Usually, this list is limited to GM events which are authorised and for which (certified) reference materials are available. If possible, different matrices (e.g. gDNA, pDNA, raw material, processed material,…) are included and different GM concentrations are used. Further the applicability of the methods is assessed by screening certified reference materials which are used in the GMOlab for validation and calibration purposes.

The **practicability** of the SYBR®Green screening methods follows directly from the fact that all methods have been developed in-house. During the development, the use of the same conditions (qPCR program, reaction volume, …) and qPCR instruments have been taken into account. This will thus allow using all methods in a same run during routine analysis of a sample.

The **specificity** of the method is first assessed *in silico* (part 4.2) and further experimentally. The screening method should be specific for the target for which it is developed and should not be homologous and give an amplification product with other sequences. The specificity is experimentally tested on all materials to which the analysis can be applied. The GM events or taxa containing the target should give a positive amplification signal, while the ones which do not contain it should give no amplification signal. An amplification signal is considered positive when a Ct value and a melting curve analysis are recorded. Absence of amplification is considered when either no Ct is recorded or when a Ct value at least 10 Ct higher than the one of the positive samples is measured. To assess the nominal Tm value, a plasmid containing the construct under analysis may be used.

As the screening methods developed and validated at the GMOlab are based on the SYBR®Green detection chemistry, the melting temperature of the amplicon is an important parameter related to the specificity of the method. The melting temperature (Tm) of a DNA sequence is dependent on a large number of factors, among which the ionic conditions in the sample solution, the DNA nature (sequence, secondary structure, etc.) and the starting concentration of the DNA molecule (Hillen et al., 1981; Rouzina & Bloomfield, 2001). Moreover different qPCR instruments tend to measure slightly different values for a given amplicon (due to differences in heating block control, mathematical integration, extrapolation, etc.). The variation of the Tm follows a normal distribution and the Tm of the method is calculated as the average Tm from all the data obtained during validation. Additionally, a Tm confidence interval is calculated (Tm ± 3 standard deviations) which is used further to decide whether the correct target has been amplified (part 6). The Tm and its confidence interval can be updated regularly by adding data from analysis of routine samples to the existing dataset.

Using the data from the *in silico* and experimental specificity tests, mostly only one primer pair is selected for determination of the method sensitivity (LOD and LOQ) and repeatability.

To assess the **sensitivity** of the developed method, a GM event containing the target is used (usually a CRM with a known GM%). It should however be noted that the GM-specific CRM are certified for the content of a specific GM event and not for the content of the screening target (promoter, coding sequence, terminator). This demonstrates that the preliminary information on the elements inserted in a GM event and their copy number is crucial in order to estimate the correct copy number of the target. For taxon-specific markers, this assessment can be done using a wild type (non-GM) material. The LOD and the LOQ are determined on basis of serial dilutions starting from at least 2000 target copies until the theoretical zero copy numbers. Each of the dilutions is run in six replicates.

The **LOD** is set up at the level where less than 5% false negatives are observed (Codex Alimentarius, 2009). As it is not feasible to perform the analysis on a large number of PCR replicates, six repeats are run per dilution point. If all six repeats are positive, this means that 95% of the time a positive sample will indeed be detected. Therefore the LOD of the screening method is set at the haploid genome copy level at which all six replicates provide a specific positive signal (n = 6; 6/6 specific signals) (AFNOR XP V 03-020-2).

The **LOQ** is defined as the target copy number with a similar positive PCR result (expressed as Ct value) upon six-fold measurement of the target sequence in the same DNA sample with a minor standard deviation (SDCt<0,5) (AFNOR XP V 03-020-2). A screening target is in principle not quantified, but the LOQ can give an idea about the content of the target in an unknown sample.

Additionally, the **precision** (inter-run repeatability) of the method is determined. In practice this is done by calculating the relative repeatability standard deviation (RSDr%) on each of the dilutions used to determine the LOD and LOQ. Hereto, the experiment is performed under repeatability conditions (in a short period of time, on the same qPCR instrument by the same operator) in four independent runs. The RSDr% is calculated according to the ISO 5725-2. The method is accepted as valid when the RSDr% is below 25%.

## **4.4 Conclusion**

384 Polymerase Chain Reaction

The in-house validation of a SYBR®Green screening method is based on the determination of several method characteristics that are required for the validation of event-specific quantitative methods (ENGL, 2008 - part 5), namely applicability, practicability, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ) and precision (RSDr%). The definitions of these parameters can be found in the glossary. The GMOlab has developed its own experimental set up in order to assess these parameters. Upon validation the results are evaluated and if they meet the acceptance criteria the method can be used under accreditation. Additionally, the critical values which are introduced in the CoSYPS (part 6)

The method is **applicable** when it detects the target in the respective GMO for which it was designed. To test this aspect of a method a list of GM events containing the target (positive samples) and events not containing the target (negative samples) is made. Usually, this list is limited to GM events which are authorised and for which (certified) reference materials are available. If possible, different matrices (e.g. gDNA, pDNA, raw material, processed material,…) are included and different GM concentrations are used. Further the applicability of the methods is assessed by screening certified reference materials which are

The **practicability** of the SYBR®Green screening methods follows directly from the fact that all methods have been developed in-house. During the development, the use of the same conditions (qPCR program, reaction volume, …) and qPCR instruments have been taken into account. This will thus allow using all methods in a same run during routine analysis of a

The **specificity** of the method is first assessed *in silico* (part 4.2) and further experimentally. The screening method should be specific for the target for which it is developed and should not be homologous and give an amplification product with other sequences. The specificity is experimentally tested on all materials to which the analysis can be applied. The GM events or taxa containing the target should give a positive amplification signal, while the ones which do not contain it should give no amplification signal. An amplification signal is considered positive when a Ct value and a melting curve analysis are recorded. Absence of amplification is considered when either no Ct is recorded or when a Ct value at least 10 Ct higher than the one of the positive samples is measured. To assess the nominal Tm value, a

As the screening methods developed and validated at the GMOlab are based on the SYBR®Green detection chemistry, the melting temperature of the amplicon is an important parameter related to the specificity of the method. The melting temperature (Tm) of a DNA sequence is dependent on a large number of factors, among which the ionic conditions in the sample solution, the DNA nature (sequence, secondary structure, etc.) and the starting concentration of the DNA molecule (Hillen et al., 1981; Rouzina & Bloomfield, 2001). Moreover different qPCR instruments tend to measure slightly different values for a given amplicon (due to differences in heating block control, mathematical integration, extrapolation, etc.). The variation of the Tm follows a normal distribution and the Tm of the method is calculated as the average Tm from all the data obtained during validation. Additionally, a Tm confidence interval is calculated (Tm ± 3 standard deviations) which is used further to decide whether the correct target has been amplified (part 6). The Tm and its

**Green screening method**

**4.3 Validation of a SYBR®**

sample.

are determined during the in-house validation.

used in the GMOlab for validation and calibration purposes.

plasmid containing the construct under analysis may be used.

As, to date, no instructions on the development and validation of screening methods are available, the laboratories need to set up their own experimental plan and criteria. At the WIV-ISP-GMOlab, development and validation of SYBR®Green methods for screening purposes is done in a harmonized way to allow applying the methods in a single qPCR run. The parameters evaluated, the way to perform this assessment and the acceptance criteria are based on previously published documents (ENGL, 2008; Codex Alimentarius, 2009; AFNOR XP V 03-020-2).

Upon evaluation of all the necessary parameters and their accordance with the set criteria, a validation dossier is established. The LOD, LOQ (expressed as a Ct value) and the Tm interval are introduced into the CoSYPS Decision Support System and serve as decision values to conclude if a sample is positive for the target or not (part 6). Subsequently the method is implemented in routine GMO detection under ISO 17025.

Development of a Molecular Platform

**quantification**

harmonised methods applicable in official GMO detection.

the world and against those still in development.

used for different GM events is needed.

applied in a routine laboratory.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 387

with the results of the study and the validated protocol. These are submitted to the European Food Safety Authority (EFSA) and are subsequently published on the EU-RL GMFF official website. Upon publication the validated methods become official methods. The method validation thus provides the enforcement laboratories with standardised and

The requirements for method **specificity** are laid down in the legislation. The method submitted has to be event-specific (based on the specific sequence of the plant-transgenic construct junction, part 3) and should detect only the specific GMO submitted for authorisation to be useful for unequivocal detection/identification/quantification of the GM event (EC/641/2004). To demonstrate that the method is event-specific, it has to be tested against all GM events from the applicant which are currently authorised in different parts of

As the submitted methods are quantitative, they also include a reference taxon-specific assay. The specificity of this assay should also be tested. For taxon-specific assays the target should be preferably a unique sequence present in a single copy in the target plant genome. The copy number and the specificity have to be assessed *in silico* by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches against known databases. In addition, the taxon-specific target should not show amplification signals with close relatives or taxa of the most important food crops. Usually, the different biotech companies develop their own taxon-specific method and test it on a range of taxa selected by them. This can pose several problems for the laboratories applying the methods. Firstly, there is no standard list of taxa and varieties to be included in the test. Ideally, the reference assay should be tested on a large range of varieties covering the existing natural variation within the taxon in order to assure that it will amplify any material from the plant species targeted by the method. Secondly, the existence of more than one reference system for events of the same plant taxon requires the use of several reference assays in quantification, which increases the costs of the analysis by the laboratory. In this context the requirements for the specificity of the taxonspecific reference assays should be made more precise and harmonisation in the methods

Information on the **applicability** of the method should be provided. This includes information on the scope of the method. In addition, information on known interferences

The **practicability** of the method should be demonstrated. For instance, methods where the reference and the event-specific assays are run on different PCR plates or under different PCR cycling conditions are less practicable and would be time and cost consuming when

Besides these criteria, other parameters related to the method performance are assessed namely the **dynamic range, linearity, amplification efficiency, LOD and LOQ, trueness, precision and robustness**. The definitions of all parameters can be found in the glossary.

with other analytes and the applicability to certain matrices should be supplied.

**5.2 Evaluated parameters for newly developed event-specific methods for GMO**

**5.2.1 Evaluation of method performance characteristics by the EURL-GMFF**

## **5. Validation of a qPCR method for GMO quantification and its implementation in a routine laboratory under ISO 17025 accreditation**

## **5.1 Introduction into the legal context**

Regulation (EC) 1829/2003 on genetically modified food and feed defines that food and feed products containing or derived from GMO must be labelled. The labelling requirements do not apply to food and feed containing GMO in a proportion not higher than 0,9% of the ingredients, provided that this presence is adventitious or technically unavoidable. Moreover, the recently adopted "Low Level Presence" Commission Regulation (EC/619/2011) requires a reliable quantification at a level of 0.1%. Member States are responsible for monitoring the GMO content of products and compliance with GMO labelling requirements. In this context, the enforcement of the EU legislation on GMO labelling requires GMO detection methods that are sound, precise and robust. It is, therefore, an essential requirement to use validated methods for GMO detection and quantification. Only in this manner it can be assured that independent control laboratories achieve comparable analysis results and are able to fulfil regulatory tasks (JRC, 2010).

The submission and validation of a GMO detection method is an integral part of the regulatory and approval process for GM food and feed to be placed on the market (EC/1829/2003). This Commission Regulation states that the application for authorisation should include, amongst others, "methods for detection, sampling and identification of the transformation event". As a consequence, the biotech companies have to provide detection protocols and control samples to validate the event-specific method to the EU-RL GMFF. These methods should be based on the real-time PCR technology (EC/787/2004). In view of the European harmonisation and standardisation of methods for sampling, detection, identification and quantification of GMO, the EU-RL has published a list of parameters to be tested and their acceptance criteria in the a document "Definition of minimum performance requirements for analytical methods of GMO testing" (ENGL, 2008).

A GM event cannot be authorised in the EU before a relevant detection method has been validated. The method validation process is conducted by the European Commission's Joint Research Centre (JRC) in its capacity as European Union Reference Laboratory for GM Food and Feed, and is assisted in its task by the European Network of GMO Laboratories. Commission Regulation EC/882/2004 establishes that analytical methods used for food and feed control must be verified by control laboratories before their use (JRC, 2010). In practice, after testing of the material and protocol, the JRC distributes the sample material and corresponding reagents to the participating laboratories in a ring trial. The validation ring trials are organised according to the requirements set up in ISO 5725 and following the IUPAC protocol (IUPAC, 1995). In such a collaborative validation trial, the EU-RL is assisted by the National Reference Laboratories (NRL) which are assigned as official control laboratories at national level (EC/882/2004). The NRL have to be accredited under ISO 17025 standard. Usually there are 12-13 participating laboratories, randomly selected from all available NRL. The validation ring trial aims at determining the method performance characteristics.

In this way the submitted method is evaluated with regard to the validation criteria. Failure to meet these criteria leads to rejection of the method and consequently to a delay in the authorisation of the GMO. Upon acceptance, the EU-RL GMFF prepares a validation report

Regulation (EC) 1829/2003 on genetically modified food and feed defines that food and feed products containing or derived from GMO must be labelled. The labelling requirements do not apply to food and feed containing GMO in a proportion not higher than 0,9% of the ingredients, provided that this presence is adventitious or technically unavoidable. Moreover, the recently adopted "Low Level Presence" Commission Regulation (EC/619/2011) requires a reliable quantification at a level of 0.1%. Member States are responsible for monitoring the GMO content of products and compliance with GMO labelling requirements. In this context, the enforcement of the EU legislation on GMO labelling requires GMO detection methods that are sound, precise and robust. It is, therefore, an essential requirement to use validated methods for GMO detection and quantification. Only in this manner it can be assured that independent control laboratories achieve comparable analysis results and are able to fulfil regulatory tasks (JRC, 2010).

The submission and validation of a GMO detection method is an integral part of the regulatory and approval process for GM food and feed to be placed on the market (EC/1829/2003). This Commission Regulation states that the application for authorisation should include, amongst others, "methods for detection, sampling and identification of the transformation event". As a consequence, the biotech companies have to provide detection protocols and control samples to validate the event-specific method to the EU-RL GMFF. These methods should be based on the real-time PCR technology (EC/787/2004). In view of the European harmonisation and standardisation of methods for sampling, detection, identification and quantification of GMO, the EU-RL has published a list of parameters to be tested and their acceptance criteria in the a document "Definition of minimum performance

A GM event cannot be authorised in the EU before a relevant detection method has been validated. The method validation process is conducted by the European Commission's Joint Research Centre (JRC) in its capacity as European Union Reference Laboratory for GM Food and Feed, and is assisted in its task by the European Network of GMO Laboratories. Commission Regulation EC/882/2004 establishes that analytical methods used for food and feed control must be verified by control laboratories before their use (JRC, 2010). In practice, after testing of the material and protocol, the JRC distributes the sample material and corresponding reagents to the participating laboratories in a ring trial. The validation ring trials are organised according to the requirements set up in ISO 5725 and following the IUPAC protocol (IUPAC, 1995). In such a collaborative validation trial, the EU-RL is assisted by the National Reference Laboratories (NRL) which are assigned as official control laboratories at national level (EC/882/2004). The NRL have to be accredited under ISO 17025 standard. Usually there are 12-13 participating laboratories, randomly selected from all available NRL. The validation ring trial aims at determining the method

In this way the submitted method is evaluated with regard to the validation criteria. Failure to meet these criteria leads to rejection of the method and consequently to a delay in the authorisation of the GMO. Upon acceptance, the EU-RL GMFF prepares a validation report

requirements for analytical methods of GMO testing" (ENGL, 2008).

**5. Validation of a qPCR method for GMO quantification and its**

**5.1 Introduction into the legal context**

performance characteristics.

**implementation in a routine laboratory under ISO 17025 accreditation**

with the results of the study and the validated protocol. These are submitted to the European Food Safety Authority (EFSA) and are subsequently published on the EU-RL GMFF official website. Upon publication the validated methods become official methods. The method validation thus provides the enforcement laboratories with standardised and harmonised methods applicable in official GMO detection.

#### **5.2 Evaluated parameters for newly developed event-specific methods for GMO quantification**

## **5.2.1 Evaluation of method performance characteristics by the EURL-GMFF**

The requirements for method **specificity** are laid down in the legislation. The method submitted has to be event-specific (based on the specific sequence of the plant-transgenic construct junction, part 3) and should detect only the specific GMO submitted for authorisation to be useful for unequivocal detection/identification/quantification of the GM event (EC/641/2004). To demonstrate that the method is event-specific, it has to be tested against all GM events from the applicant which are currently authorised in different parts of the world and against those still in development.

As the submitted methods are quantitative, they also include a reference taxon-specific assay. The specificity of this assay should also be tested. For taxon-specific assays the target should be preferably a unique sequence present in a single copy in the target plant genome. The copy number and the specificity have to be assessed *in silico* by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches against known databases. In addition, the taxon-specific target should not show amplification signals with close relatives or taxa of the most important food crops. Usually, the different biotech companies develop their own taxon-specific method and test it on a range of taxa selected by them. This can pose several problems for the laboratories applying the methods. Firstly, there is no standard list of taxa and varieties to be included in the test. Ideally, the reference assay should be tested on a large range of varieties covering the existing natural variation within the taxon in order to assure that it will amplify any material from the plant species targeted by the method. Secondly, the existence of more than one reference system for events of the same plant taxon requires the use of several reference assays in quantification, which increases the costs of the analysis by the laboratory. In this context the requirements for the specificity of the taxonspecific reference assays should be made more precise and harmonisation in the methods used for different GM events is needed.

Information on the **applicability** of the method should be provided. This includes information on the scope of the method. In addition, information on known interferences with other analytes and the applicability to certain matrices should be supplied.

The **practicability** of the method should be demonstrated. For instance, methods where the reference and the event-specific assays are run on different PCR plates or under different PCR cycling conditions are less practicable and would be time and cost consuming when applied in a routine laboratory.

Besides these criteria, other parameters related to the method performance are assessed namely the **dynamic range, linearity, amplification efficiency, LOD and LOQ, trueness, precision and robustness**. The definitions of all parameters can be found in the glossary.

Development of a Molecular Platform

**5.4 Conclusion**

quantifiable.

**6.1 General strategy**

range of (-2;2) should have been obtained.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 389

not deviate more than 25% from the true value. In the case of a PT sample a z-score in the

The **Relative Repeatability Standard Deviation (RSDr)** should be calculated on at least 16 single test results obtained under repeatability conditions. Repeatability should be available

Furthermore, the enforcement laboratory should estimate the **sensitivity** of the method. Hereto, four parameters can be calculated. The *Relative LOQ (LOQrel)* is estimated at low concentration(s) of positive material e.g. 0,1%. The LOQrel is set at this level if the RSDr is below 25%. The *Absolute LOQ (LOQabs)* is estimated by measuring dilution series of low copy numbers of the target. The LOQabs is set as the last dilution where the RSDr is lower than 25%. The *Relative LOD (LODrel)* is estimated using ten replicates of a positive control material with a low GM level. The LODrel is set at this level if the ten replicates show a positive amplification. The *Absolute LOD (LODabs)* is estimated as the copy number at which not more than 5% false negatives are obtained. In practice this is performed by evaluating ten PCR replicates of low copy number of the target. The LODabs is set at this level if the ten replicates score positive.

A GMO quantification method filed by the biotech companies together with the application for authorisation follows different steps. Firstly, the developer needs to provide information on the performance of the method. Hereto, he needs to evaluate different parameters as laid down in the ENGL document (ENGL, 2008). Secondly, the EU-RL GMFF evaluates the submitted information and decides whether the dossier is in compliance with the set criteria. Thirdly, the EU-RL organises a ring trial to validate the method. Hereto it gets the support of the different NRL that participate in the validation. Fourthly, the enforcement laboratories need to assess a number of parameters before to implement the method in their

At WIV-ISP-GMOlab, the assessed parameters and the data obtained during the in-house verification are gathered in a validation dossier. The event-specific method is in a first time used as a qualitative identification method in the second step of GMO analysis. The critical parameters determined during the in-house validation for these methods are the LODabs and LOQabs. These parameters, expressed as Ct values, are introduced into the DSS and serve as a threshold to decide if the GM event is present in the sample and in case of presence if it is

For quantification methods, no real DSS exists but different parameters are evaluated at each use in routine analysis and have to be in compliance with the set criteria. In a first step, the parameters of the calibration curves of the event-specific and the taxon-specific method (linearity, slope, PCR efficiency) are evaluated. Additionally, control samples (0,1% and 1%) are quantified and the result has to fulfil the acceptance criterion for trueness. In this way

**6. Introduction of the qPCR methods in the Decision Support System (DSS)**

As described before (part 3), to cover the broadest GMO spectra, SYBR®Green qPCR methods have been developed and validated in the GMO detection platform. In this context,

laboratory for routine analysis under ISO 17025 accreditation.

the obtained quantitative results for unknown samples are validated.

for all tested GM levels. The RSDr needs to be equal or below 25% to be acceptable.

#### **5.2.2 Evaluation of method performance characteristics, performed by the analysis of the results of the inter-laboratory collaborative trial**

Once the EU-RL GMFF has made a scientific evaluation of the method based on the performance of the above-mentioned parameters (as provided by the method developer), it organizes a validation ring trial (concerning dynamic range, precision, relative reproducibility standard deviation and trueness). The participating laboratories receive the necessary samples and reagents and a detailed experimental protocol. It should be noted that the purpose of the ring trial is to assess the performance of the method and not of the laboratory. Therefore each participant has to follow the experimental procedure strictly. The results obtained by the laboratories are expressed as GM% for each tested level. These results are further scrutinised for outliers by the EU-RL GMFF using statistical methods recommended by ISO 5725. In addition, the mean value is calculated for each GM level analysed. Based on the parameters assessed during the ring trial, a conclusion is made on the compliance of the method with the ENGL method acceptance criteria and if it can be considered applicable in regard to the requirements of EC/641/2004.

### **5.3 Implementation of a validated event-specific method in a testing laboratory**

When the interlaboratory validation study is completed and the method is considered as applicable, the method is ready to be implemented in routine testing laboratories like the GMOlab.

On the one hand, Commission Regulation EC/882/2004 states that official laboratories shall be accredited according to the ISO 17025 standard. An ISO 17025 accreditation, under a fixed or flexible scope, implies that "the laboratory shall confirm that it can properly operate standard methods before introducing the tests for calibrations". On the other hand, according to the same regulation, it is the task of the EU-RL GMFF to provide the NRL with details of analytical methods, including reference methods. In this context, guidelines for implementation of the validated methods in the routine laboratory are set up by the ENGL in the document "Verification of analytical methods for GMO testing when implementing interlaboratory validated methods" (ENGL, 2011). These guidelines reflect the requirements set up in the document "Definition of the Minimum Performance Requirements for analytical methods of GMO testing" (ENGL, 2008), but also give additional guidance on how to design the experimental set up and to calculate the required values. In practice the laboratories have to design the quantification experiment in which two or three GM levels are quantified and the parameters described hereunder have to be assessed.

**Dynamic range, R2 coefficient and amplification efficiency:** these parameters can be calculated simultaneously from calibration curves when testing other parameters (trueness and precision). For each target, the average values of at least two calibration curves should be taken. The dynamic range should be tested between 1/10th of the threshold value and 5 times this value i.e. between 0,09% and 4,5% for the 0,9% labelling threshold. The PCR efficiency should be between 90 and 100% and the R2 coefficient needs to be equal or above 0,98 to have a linear curve.

**Trueness** should be determined at a level close to the level set in the legislation (0,9%) or according to the intended use of the method and additionally at a level close to the LOQ. The trueness can be measured using a CRM or if not available on a sample from a proficiency test (PT). To comply with the acceptance criterion, the measured value should not deviate more than 25% from the true value. In the case of a PT sample a z-score in the range of (-2;2) should have been obtained.

The **Relative Repeatability Standard Deviation (RSDr)** should be calculated on at least 16 single test results obtained under repeatability conditions. Repeatability should be available for all tested GM levels. The RSDr needs to be equal or below 25% to be acceptable.

Furthermore, the enforcement laboratory should estimate the **sensitivity** of the method. Hereto, four parameters can be calculated. The *Relative LOQ (LOQrel)* is estimated at low concentration(s) of positive material e.g. 0,1%. The LOQrel is set at this level if the RSDr is below 25%. The *Absolute LOQ (LOQabs)* is estimated by measuring dilution series of low copy numbers of the target. The LOQabs is set as the last dilution where the RSDr is lower than 25%.

The *Relative LOD (LODrel)* is estimated using ten replicates of a positive control material with a low GM level. The LODrel is set at this level if the ten replicates show a positive amplification. The *Absolute LOD (LODabs)* is estimated as the copy number at which not more than 5% false negatives are obtained. In practice this is performed by evaluating ten PCR replicates of low copy number of the target. The LODabs is set at this level if the ten replicates score positive.

## **5.4 Conclusion**

388 Polymerase Chain Reaction

**5.2.2 Evaluation of method performance characteristics, performed by the analysis of**

Once the EU-RL GMFF has made a scientific evaluation of the method based on the performance of the above-mentioned parameters (as provided by the method developer), it organizes a validation ring trial (concerning dynamic range, precision, relative reproducibility standard deviation and trueness). The participating laboratories receive the necessary samples and reagents and a detailed experimental protocol. It should be noted that the purpose of the ring trial is to assess the performance of the method and not of the laboratory. Therefore each participant has to follow the experimental procedure strictly. The results obtained by the laboratories are expressed as GM% for each tested level. These results are further scrutinised for outliers by the EU-RL GMFF using statistical methods recommended by ISO 5725. In addition, the mean value is calculated for each GM level analysed. Based on the parameters assessed during the ring trial, a conclusion is made on the compliance of the method with the ENGL method acceptance criteria and if it can be

**the results of the inter-laboratory collaborative trial**

considered applicable in regard to the requirements of EC/641/2004.

GMOlab.

0,98 to have a linear curve.

**5.3 Implementation of a validated event-specific method in a testing laboratory**

are quantified and the parameters described hereunder have to be assessed.

When the interlaboratory validation study is completed and the method is considered as applicable, the method is ready to be implemented in routine testing laboratories like the

On the one hand, Commission Regulation EC/882/2004 states that official laboratories shall be accredited according to the ISO 17025 standard. An ISO 17025 accreditation, under a fixed or flexible scope, implies that "the laboratory shall confirm that it can properly operate standard methods before introducing the tests for calibrations". On the other hand, according to the same regulation, it is the task of the EU-RL GMFF to provide the NRL with details of analytical methods, including reference methods. In this context, guidelines for implementation of the validated methods in the routine laboratory are set up by the ENGL in the document "Verification of analytical methods for GMO testing when implementing interlaboratory validated methods" (ENGL, 2011). These guidelines reflect the requirements set up in the document "Definition of the Minimum Performance Requirements for analytical methods of GMO testing" (ENGL, 2008), but also give additional guidance on how to design the experimental set up and to calculate the required values. In practice the laboratories have to design the quantification experiment in which two or three GM levels

**Dynamic range, R2 coefficient and amplification efficiency:** these parameters can be calculated simultaneously from calibration curves when testing other parameters (trueness and precision). For each target, the average values of at least two calibration curves should be taken. The dynamic range should be tested between 1/10th of the threshold value and 5 times this value i.e. between 0,09% and 4,5% for the 0,9% labelling threshold. The PCR efficiency should be between 90 and 100% and the R2 coefficient needs to be equal or above

**Trueness** should be determined at a level close to the level set in the legislation (0,9%) or according to the intended use of the method and additionally at a level close to the LOQ. The trueness can be measured using a CRM or if not available on a sample from a proficiency test (PT). To comply with the acceptance criterion, the measured value should A GMO quantification method filed by the biotech companies together with the application for authorisation follows different steps. Firstly, the developer needs to provide information on the performance of the method. Hereto, he needs to evaluate different parameters as laid down in the ENGL document (ENGL, 2008). Secondly, the EU-RL GMFF evaluates the submitted information and decides whether the dossier is in compliance with the set criteria. Thirdly, the EU-RL organises a ring trial to validate the method. Hereto it gets the support of the different NRL that participate in the validation. Fourthly, the enforcement laboratories need to assess a number of parameters before to implement the method in their laboratory for routine analysis under ISO 17025 accreditation.

At WIV-ISP-GMOlab, the assessed parameters and the data obtained during the in-house verification are gathered in a validation dossier. The event-specific method is in a first time used as a qualitative identification method in the second step of GMO analysis. The critical parameters determined during the in-house validation for these methods are the LODabs and LOQabs. These parameters, expressed as Ct values, are introduced into the DSS and serve as a threshold to decide if the GM event is present in the sample and in case of presence if it is quantifiable.

For quantification methods, no real DSS exists but different parameters are evaluated at each use in routine analysis and have to be in compliance with the set criteria. In a first step, the parameters of the calibration curves of the event-specific and the taxon-specific method (linearity, slope, PCR efficiency) are evaluated. Additionally, control samples (0,1% and 1%) are quantified and the result has to fulfil the acceptance criterion for trueness. In this way the obtained quantitative results for unknown samples are validated.

## **6. Introduction of the qPCR methods in the Decision Support System (DSS)**

#### **6.1 General strategy**

As described before (part 3), to cover the broadest GMO spectra, SYBR®Green qPCR methods have been developed and validated in the GMO detection platform. In this context,

Development of a Molecular Platform

present.

an integer).

**interpretation**

crl.jrc.ec.europa.eu).

conditions (part 4).

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 391

multiple targets are present in a GMO and the CoSYPS contains methods for each of these targets, all targets present in that GMO must be positive to conclude that this GMO might be

The second step in the CoSYPS algorithm is based on a mathematical model. A unique prime number (a prime number is a natural number that has exactly two distinct natural number divisors: 1 and itself) is associated with each particular screening method. When the sample is considered positive for a certain screening element, this specific prime number is assigned to the sample. When it is considered negative, the number 1 (neutral element in multiplication) is assigned. By multiplying all assigned values, the algorithm calculates the "Gödel prime product" (GPPsample) of the sample (the product of the prime numbers corresponding to the positive scoring screening methods). In a similar way each GMO can be represented by a product of the different prime numbers corresponding to the elements belonging to the GMO. This product is designed as the "Gödel prime product" (GPPGMO) of the GMO and represents a "mathematical tag" for this GMO. Note that several GMO can be

associated with a same GPP product as they comprise the same genetic elements.

potential GMO present in the sample by a series of simple divisions.

previously introduced in the Decision Support System.

The third step of the CoSYPS is based on the fact that, as a consequence of the nature of prime numbers, the division of the GPP by any of the prime numbers used in the generation of the GPP is an integer. Therefore the presence of a target in a GMO can be mathematically traced by generating this fraction: the program makes the ratio between the GPPsample and the GPPGMO to identify which GMO could be present in the sample (the division generates

Consequently, on the basis of the positive signal(s) obtained during the screening for each specific SYBR®Green qPCR method, the specific prime number assigned to each method is scored by the CoSYPS. The multiplication of these prime numbers allows the CoSYPS to calculate the GPP for the analysed sample. From this number, the CoSYPS can select all the

On the basis of outcome of the CoSYPS analysis a set of candidate GMO which could possibly reside within the product can be identified. In order to confirm the presence of a certain GM event in this product, event-specific Taqman® qPCR analysis is performed in a next step by applying methods validated and published by the EU-RL (http://gmo-

During the sample analysis, the Ct value obtained as outcome of the event-specific qPCR is recorded. This Ct value is compared to the LOD and LOQ (as determined during the verification of the identification method in the laboratory - part 5). These values were

A GM event is considered detectable by the DSS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained. The LOD was obtained under repeatability

To conclude which GM events are effectively present and identified in the sample, the DSS retains all prime numbers of the GM event with a Ct value below the Ct value of the LOD (+

**6.3 Integration of an event-specific method in the Decision Support System and**

it rapidly becomes tedious in routine analyses to manually combine all the screening results in order to decide which GMO are potentially present in a sample. Therefore, in support to the qPCR data, a simple mathematical model has been developed to automatically calculate the possible presences in a product based on the outcome of the qPCR screening analysis (Van den Bulcke et al., 2010). The CoSYPS, standing for Combinatory SYBR®Green qPCR screening, represents a novel tool for GMO analysis based on the SYBR®Green qPCR technology. Using this decision support system alone is not sufficient. The suspected GM events need to be specifically identified in a second step, using e.g. the EU-RL Taqman® event-specific qPCR method(s). In a third step, the positively identified GM events are quantified to asses if their content complies or not with the 0,9% labelling threshold (EC1830/2003).

This newly developed tool is a versatile, cost-effective and time-efficient approach in assessing the GMO presence in analytical samples and can be applied in routine analysis for enforcement purposes. The full system has been patent protected (Van den Bulcke et al., 2008).

Here the construction, functioning and the theoretical basis of the CoSYPS will be described. Further explanation on the mathematical functioning of the CoSYPS may be found in the recently published paper "A theoretical introduction to "Combinatory SYBR®Green qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants" (Van den Bulcke et al., 2010).

## **6.2 Screening for GMO candidates by CoSYPS analysis**

The CoSYPS is based on the determination of the presence of certain element(s) originating from GMO and plant taxa frequently occurring in food and feed products. Hereto, SYBR®Green qPCR analysis of gDNA extracted from the product is performed, using primer pairs targeting different (multiple) discriminatory marker amplicons (part 3 and table 2).

During the SYBR®Green qPCR analysis of the sample, two critical qPCR parameters are recorded for each method used: the Ct and Tm values. Within the Decision Support System the obtained values are then compared to the LOD (expressed as a Ct value – see glossary) determined in the validation of the qPCR screening method and the nominal Tm value of the amplicon (see glossary). Both parameters are used as decision criteria for the analysis and are incorporated as such in the CoSYPS Decision Support System.

In a first step, the CoSYPS algorithm compares the measured Ct and the Tm values for each screening element with the corresponding "decision values" in the DSS. The latter values are determined during the in-house validation of the method (part 4). A signal generated in SYBR®Green qPCR analysis for a sample is considered as positive by the CoSYPS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained and the amplicon has a Tm value that falls within the determined Tm confidence interval (part 4). In agreement with the decision principles of the ISO norm 24276 (twice positive, twice negative), all decisions within the CoSYPS are based on the extraction and analysis of two distinct representative sub-extracts and eventually confirmed by a third analysis in case of ambiguous results (one positive, one negative). Therefore, a sample is positive for a specific screening element when the Ct and Tm results are unambiguously for both sub-extracts. Any positive signal obtained with a SYBR®Green qPCR method targeting a particular GM element indicates that a GMO comprising this target could be present in the sample. When several GMO contain the same target, a positive result generated by this screening method indicates that potentially all these GMO may be present in the sample. However, when

it rapidly becomes tedious in routine analyses to manually combine all the screening results in order to decide which GMO are potentially present in a sample. Therefore, in support to the qPCR data, a simple mathematical model has been developed to automatically calculate the possible presences in a product based on the outcome of the qPCR screening analysis (Van den Bulcke et al., 2010). The CoSYPS, standing for Combinatory SYBR®Green qPCR screening, represents a novel tool for GMO analysis based on the SYBR®Green qPCR technology. Using this decision support system alone is not sufficient. The suspected GM events need to be specifically identified in a second step, using e.g. the EU-RL Taqman® event-specific qPCR method(s). In a third step, the positively identified GM events are quantified to asses if their

This newly developed tool is a versatile, cost-effective and time-efficient approach in assessing the GMO presence in analytical samples and can be applied in routine analysis for enforcement purposes. The full system has been patent protected (Van den Bulcke et al., 2008). Here the construction, functioning and the theoretical basis of the CoSYPS will be described. Further explanation on the mathematical functioning of the CoSYPS may be found in the recently published paper "A theoretical introduction to "Combinatory SYBR®Green qPCR screening", a matrix-based approach for the detection of materials derived from genetically

The CoSYPS is based on the determination of the presence of certain element(s) originating from GMO and plant taxa frequently occurring in food and feed products. Hereto, SYBR®Green qPCR analysis of gDNA extracted from the product is performed, using primer pairs targeting different (multiple) discriminatory marker amplicons (part 3 and table 2).

During the SYBR®Green qPCR analysis of the sample, two critical qPCR parameters are recorded for each method used: the Ct and Tm values. Within the Decision Support System the obtained values are then compared to the LOD (expressed as a Ct value – see glossary) determined in the validation of the qPCR screening method and the nominal Tm value of the amplicon (see glossary). Both parameters are used as decision criteria for the analysis and

In a first step, the CoSYPS algorithm compares the measured Ct and the Tm values for each screening element with the corresponding "decision values" in the DSS. The latter values are determined during the in-house validation of the method (part 4). A signal generated in SYBR®Green qPCR analysis for a sample is considered as positive by the CoSYPS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained and the amplicon has a Tm value that falls within the determined Tm confidence interval (part 4). In agreement with the decision principles of the ISO norm 24276 (twice positive, twice negative), all decisions within the CoSYPS are based on the extraction and analysis of two distinct representative sub-extracts and eventually confirmed by a third analysis in case of ambiguous results (one positive, one negative). Therefore, a sample is positive for a specific screening element when the Ct and Tm results are unambiguously for both sub-extracts. Any positive signal obtained with a SYBR®Green qPCR method targeting a particular GM element indicates that a GMO comprising this target could be present in the sample. When several GMO contain the same target, a positive result generated by this screening method indicates that potentially all these GMO may be present in the sample. However, when

content complies or not with the 0,9% labelling threshold (EC1830/2003).

modified plants" (Van den Bulcke et al., 2010).

**6.2 Screening for GMO candidates by CoSYPS analysis**

are incorporated as such in the CoSYPS Decision Support System.

multiple targets are present in a GMO and the CoSYPS contains methods for each of these targets, all targets present in that GMO must be positive to conclude that this GMO might be present.

The second step in the CoSYPS algorithm is based on a mathematical model. A unique prime number (a prime number is a natural number that has exactly two distinct natural number divisors: 1 and itself) is associated with each particular screening method. When the sample is considered positive for a certain screening element, this specific prime number is assigned to the sample. When it is considered negative, the number 1 (neutral element in multiplication) is assigned. By multiplying all assigned values, the algorithm calculates the "Gödel prime product" (GPPsample) of the sample (the product of the prime numbers corresponding to the positive scoring screening methods). In a similar way each GMO can be represented by a product of the different prime numbers corresponding to the elements belonging to the GMO. This product is designed as the "Gödel prime product" (GPPGMO) of the GMO and represents a "mathematical tag" for this GMO. Note that several GMO can be associated with a same GPP product as they comprise the same genetic elements.

The third step of the CoSYPS is based on the fact that, as a consequence of the nature of prime numbers, the division of the GPP by any of the prime numbers used in the generation of the GPP is an integer. Therefore the presence of a target in a GMO can be mathematically traced by generating this fraction: the program makes the ratio between the GPPsample and the GPPGMO to identify which GMO could be present in the sample (the division generates an integer).

Consequently, on the basis of the positive signal(s) obtained during the screening for each specific SYBR®Green qPCR method, the specific prime number assigned to each method is scored by the CoSYPS. The multiplication of these prime numbers allows the CoSYPS to calculate the GPP for the analysed sample. From this number, the CoSYPS can select all the potential GMO present in the sample by a series of simple divisions.

#### **6.3 Integration of an event-specific method in the Decision Support System and interpretation**

On the basis of outcome of the CoSYPS analysis a set of candidate GMO which could possibly reside within the product can be identified. In order to confirm the presence of a certain GM event in this product, event-specific Taqman® qPCR analysis is performed in a next step by applying methods validated and published by the EU-RL (http://gmocrl.jrc.ec.europa.eu).

During the sample analysis, the Ct value obtained as outcome of the event-specific qPCR is recorded. This Ct value is compared to the LOD and LOQ (as determined during the verification of the identification method in the laboratory - part 5). These values were previously introduced in the Decision Support System.

A GM event is considered detectable by the DSS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained. The LOD was obtained under repeatability conditions (part 4).

To conclude which GM events are effectively present and identified in the sample, the DSS retains all prime numbers of the GM event with a Ct value below the Ct value of the LOD (+

Development of a Molecular Platform

Screening methods

Prime numbers

*+ 1 Ct* Subsample

Subsample

*Taqman*

*Results Present*

**6.5 Conclusion**

*Quantifiable*

cost-saving tool in GMO detection.

1

2

a. Accredited SYBR®Green qPCR available

b. CoSYPS algorithm and screening methods

GPP 1057485

*CoSYPS step 2 - Calculation of the Gödel prime product of the sample*

*CoSYPS step 3 - Assessment of potential GMO present in the sample*  GMO GPP of GMO GPPsample/ GPPGMO Decision

c. DSS and confirmation by event-specific Taqman method

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 393

*CoSYPs step1 - Sample analysis "+" is assigned when value < LOD+1 Ct; " –" is assigned when value > LOD*

Product of 3 1 11 5 13 17 1 1 29 1 1

MON810 5655 187 Confirmation by event-specific Taqman method T25 16269 65 Confirmation by event-specific Taqman method GA21 145 7293 Confirmation by event-specific Taqman method GTS 40-3-2 8835 119,69 No confirmation by event-specific Taqman

MON 810 T25 GTS 40-3-2 GA21 Subsample 1 LOD > Ct < LOQ LOD > Ct > LOQ LOD < Ct > LOQ

Subsample 2 LOD > Ct < LOQ LOD > Ct > LOQ LOD < Ct > LOQ

Table 3. Mathematical functioning of the CoSYPS, allowing demonstrating the possible presence of a set of GMO in a product based on the outcome of a qPCR screening analysis.

By combining the results of the screening analysis, the CoSYPS allows to decide in a fast way which GM events are possibly present in the sample under analysis. The use of the mathematical algorithm, which compares the GPPsample and GPPGMO, excludes the need for manual calculations and comparisons. The only thing that needs to be done by the operator is the preliminary introduction of the critical values (Ct corresponding to the LOD and LOQ, Tm) obtained during method validation in the system. Further, in identification, the obtained results for a sample are compared with the LOD and LOQ values determined during inhouse validation of the event-specific methods. From this comparison, the Decision Support System will indicate which GM events are present and at which level and thus allow deciding which GMO needs to be quantified in the sample. This Decision Support System, developed and patented by the WIV-ISP-GMOlab is thus a very efficient, user friendly and

p35S pNOS t35S tNOS CryIAb PAT/pat PAT/bar CP4 ADH1 LEC CRU

3 7 11 5 13 17 23 19 29 31 37

+ - + + + + - - + - -

+ - + + + + - - + - -

method

*Present Not detectable*

1 Ct) threshold level. The Ct value is also compared with the LOQ + 1 Ct to decide if the GM event is present at a quantifiable level.

If no authorised GMO can explain the presence of a set of screening targets, it can be concluded that the sample contains one or more unassigned targets. The unassigned signals are mostly due to unauthorised GMO or donor organisms (bacterial, viral and plant sources of transgenic elements). In such cases more complex analysis like DNA walking, DNA sequencing has to be performed outside of the routine to elucidate their origin.

## **6.4 Practical case**

As an example, the accredited SYBR®Green qPCR methods available in a qPCR platform for GMO detection and their associated prime numbers are p35S, tNOS, pNOS, t35S, CryIAb, PAT/pat, CP4, PAT/bar for the transgenic elements and ADH1, LEC and CRU for the taxon-specific markers (table 3a). The elements targeted by these methods can be found in part 3 table 2.

During the screening analysis a positive signal (correct Tm and Ct < Ct of LOD + 1 Ct) is found for the p35S, tNOS, CryIAb, t35S , PAT/pat and ADH1 elements while no positive signal was obtained for pNOS, CP4, PAT/bar and the other species-specific targets (table 3b–step1). For each positive screening marker (p35S, tNOS, CryIAb, t35S and PAT/pat and ADH1) the specific prime number is assigned to each of the corresponding methods. As the pNOS, CP4, PAT/bar targets and the other taxon-specific markers are considered as negative the assigned number for all of these methods is 1. The "Gödel prime product" of the sample (= 1057485) is calculated by multiplying all the assigned prime numbers (table 3b-step2). The CoSYPS will compare this GPPsample with the GPP of all GM events that have previously been introduced in the system. The example is given here for four GM events.

The transgenic MON 810 and T25 events are described as a function of three transgenic elements (p35S, tNOS, CryIAb) and (p35S, t35S, PAT/pat) respectively and one maizespecific (ADH1). The GA21 maize is covered by the tNOS and maize–specific element. The GTS40-3-2 event is defined by three transgenic elements (p35S, tNOS, CP4) and the soybean endogen (LEC). Consequently, the "Gödel prime product" of the MON 810, T25, GA21 and GTS40-3-2 are 5655 (= 3 X 5 X 13 X 29), 16269 (= 3 X 11 X17 X 29), 145 (= 5 X 29) and 8835 (= 3 X 5 X 19 X 31) respectively (table 3b-step 3).

To assess which GMO are potentially present in the sample, the "Gödel prime product" of the sample is divided by the GPP of each GMO (table 3b-step 3). The result is an integer only for MON 810, T25 and GA21. From the screening analysis, the CoSYPS thus predicts that MON 810, T25 and GA21 are potentially present while GTS40-3-2 is not. As a consequence MON 810, GA21 and T25 have to be further analysed with the event-specific method to confirm their presence.

In order to confirm the presence of MON 810, T25 and GA21 in the sample product, the event-specific qPCR analyses are performed. The results (expressed as Ct values) confirm the presence of MON 810 and T25 while GA21 is not detectable (table 3c). The Ct values obtained are compared with the LOQ + 1 Ct of each method and show that only MON 810 can be quantified. Finally the GM% of this event will be compared to the labelling threshold (0,9% mass per ingredient) in order to conclude on the conformity of the sample.

#### Development of a Molecular Platform for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 393

#### a. Accredited SYBR®Green qPCR available

392 Polymerase Chain Reaction

1 Ct) threshold level. The Ct value is also compared with the LOQ + 1 Ct to decide if the GM

If no authorised GMO can explain the presence of a set of screening targets, it can be concluded that the sample contains one or more unassigned targets. The unassigned signals are mostly due to unauthorised GMO or donor organisms (bacterial, viral and plant sources of transgenic elements). In such cases more complex analysis like DNA walking, DNA

As an example, the accredited SYBR®Green qPCR methods available in a qPCR platform for GMO detection and their associated prime numbers are p35S, tNOS, pNOS, t35S, CryIAb, PAT/pat, CP4, PAT/bar for the transgenic elements and ADH1, LEC and CRU for the taxon-specific markers (table 3a). The elements targeted by these methods can be found in

During the screening analysis a positive signal (correct Tm and Ct < Ct of LOD + 1 Ct) is found for the p35S, tNOS, CryIAb, t35S , PAT/pat and ADH1 elements while no positive signal was obtained for pNOS, CP4, PAT/bar and the other species-specific targets (table 3b–step1). For each positive screening marker (p35S, tNOS, CryIAb, t35S and PAT/pat and ADH1) the specific prime number is assigned to each of the corresponding methods. As the pNOS, CP4, PAT/bar targets and the other taxon-specific markers are considered as negative the assigned number for all of these methods is 1. The "Gödel prime product" of the sample (= 1057485) is calculated by multiplying all the assigned prime numbers (table 3b-step2). The CoSYPS will compare this GPPsample with the GPP of all GM events that have previously been introduced in the system. The example is given here for four GM events.

The transgenic MON 810 and T25 events are described as a function of three transgenic elements (p35S, tNOS, CryIAb) and (p35S, t35S, PAT/pat) respectively and one maizespecific (ADH1). The GA21 maize is covered by the tNOS and maize–specific element. The GTS40-3-2 event is defined by three transgenic elements (p35S, tNOS, CP4) and the soybean endogen (LEC). Consequently, the "Gödel prime product" of the MON 810, T25, GA21 and GTS40-3-2 are 5655 (= 3 X 5 X 13 X 29), 16269 (= 3 X 11 X17 X 29), 145 (= 5 X 29) and 8835 (= 3

To assess which GMO are potentially present in the sample, the "Gödel prime product" of the sample is divided by the GPP of each GMO (table 3b-step 3). The result is an integer only for MON 810, T25 and GA21. From the screening analysis, the CoSYPS thus predicts that MON 810, T25 and GA21 are potentially present while GTS40-3-2 is not. As a consequence MON 810, GA21 and T25 have to be further analysed with the event-specific method to

In order to confirm the presence of MON 810, T25 and GA21 in the sample product, the event-specific qPCR analyses are performed. The results (expressed as Ct values) confirm the presence of MON 810 and T25 while GA21 is not detectable (table 3c). The Ct values obtained are compared with the LOQ + 1 Ct of each method and show that only MON 810 can be quantified. Finally the GM% of this event will be compared to the labelling threshold

(0,9% mass per ingredient) in order to conclude on the conformity of the sample.

sequencing has to be performed outside of the routine to elucidate their origin.

event is present at a quantifiable level.

X 5 X 19 X 31) respectively (table 3b-step 3).

confirm their presence.

**6.4 Practical case**

part 3 table 2.

#### b. CoSYPS algorithm and screening methods

c. DSS and confirmation by event-specific Taqman method

Table 3. Mathematical functioning of the CoSYPS, allowing demonstrating the possible presence of a set of GMO in a product based on the outcome of a qPCR screening analysis.

#### **6.5 Conclusion**

By combining the results of the screening analysis, the CoSYPS allows to decide in a fast way which GM events are possibly present in the sample under analysis. The use of the mathematical algorithm, which compares the GPPsample and GPPGMO, excludes the need for manual calculations and comparisons. The only thing that needs to be done by the operator is the preliminary introduction of the critical values (Ct corresponding to the LOD and LOQ, Tm) obtained during method validation in the system. Further, in identification, the obtained results for a sample are compared with the LOD and LOQ values determined during inhouse validation of the event-specific methods. From this comparison, the Decision Support System will indicate which GM events are present and at which level and thus allow deciding which GMO needs to be quantified in the sample. This Decision Support System, developed and patented by the WIV-ISP-GMOlab is thus a very efficient, user friendly and cost-saving tool in GMO detection.

Development of a Molecular Platform

our team.

**8. Glossary**

**Applicability**

**Amplification Efficiency**

by the following equation:

method can be applied.

metrological traceability.

regression analysis. **Dynamic Range**

**Correlation Coefficient (R2)**

**Certified Reference Material (CRM)**

**European Food Safety Authority (EFSA)**

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 395

broad range of GMO and UGM and thus to improve the resolution of the system. Particular importance will be given to their use in a modular approach associated with a decision tree cascade. Moreover, our strategy aiming at developing harmonised SYBR®Green qPCR screening methods incorporated in the Combinatory SYBR®Green qPCR Screening (CoSYPS) system has a potential to be applied in other scientific fields than GMO detection. The application of this strategy for food borne pathogenic bacteria is now under development in

The amplification efficiency is the rate of amplification that leads to a theoretical slope of – 3,32 with an efficiency of 100% in each cycle. The efficiency of the reaction can be calculated

10 1 *slope Efficiency*

Applicability is the description of analytes, matrices and concentrations to which the

A Certified Reference Material is a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of

The R2 coefficient is the correlation coefficient of a (calibration) curve obtained by linear

The dynamic range is the range of concentrations over which the method performs in a

EFSA is an agency of the EU that provides independent scientific advice and

The European Network of GMO Laboratories is a platform of EU experts that plays an eminent role in the development, harmonisation and standardisation of means and methods for sampling, detection, identification and quantification of Genetically Modified Organisms (GMO) or derived products in a wide variety of matrices, covering seeds, grains, food, feed and environmental samples. The network was inaugurated in Brussels on December

linear manner with an acceptable level of trueness and precision.

communication on all matters concerning food and feed safety.

**European Network of GMO Laboratories (ENGL)**

1

<sup>−</sup> = − (1)

## **7. Conclusion**

In the near future, the number and the diversity of GM crops will continue to increase, as well as the requests for authorisation for their import for food and feed in the EU. Beside the notifications of GM events produced by multinational biotech companies, many GM events will be developed by universities, national research centres and small private companies. Thus, the chance for accidental occurrence of unapproved GMO in the EU food and feed chain trough importation will be higher. As the EU's general policy supports strong commitment to consumer protection and freedom of choice, and therefore mandatory product labelling, the development of sensitive, reliable but also cost-effective and flexible strategies for the detection of GMO in products through establishment of molecular platforms will become more and more crucial.

The GMO detection platform developed at WIV-ISP consists of a pre-PCR step namely DNA extraction and three consecutive qPCR phases. In this view, the choice of efficient methods to extract good quality DNA, in particular for processed food and feed, is a critical factor. A pre-PCR evaluation of the extracted gDNA is necessary as well as setting criteria for the purity and integrity of the DNA. Furthermore, the presence of PCR inhibitors is a major obstacle for efficient amplification in qPCR. This step may even become more important as the number of GM plant taxa becomes larger. Developing simple standard methods for genomic DNA extraction minimizing inhibition will therefore be the key for providing concordant results when using qPCR techniques.

Due to the broad range of GMO that my occur in the EU food and feed chains, the use of screening strategies only based on the 35S promoter of the Cauliflower Mosaic Virus (p35S) and the nopaline synthase terminator of *Agrobacterium tumefaciens* (tNOS) followed by the analysis of the sample with event-specific EU validated methods by the enforcement laboratories will become insufficient. As a consequence, new methods focusing on an intensive screening analysis need to be developed.

At the present time several high-tech strategies like multiplex PCR and consecutive detection and identification of the amplification products using micro-arrays (Chaouachi et al., 2008, Morisset et al., 2008, Hamels et al., 2009) or PCR combined with capillary electrophoresis (Nadal et al., 2009) have been proposed to deal with this discriminative problem and the broad diversity of GMO. However, at the present time, these technologies require additional costly equipment and investments in technical support. Furthermore, they need technological optimalisation as they show a high background at low target level. These difficulties make them less suitable for routine or enforcement purposes.

Contrary to the above-mentioned technically complex strategies, our approach based on numerous singleplex qPCR-based methods developed to function under the same reaction conditions combined with the informatics decision support tool CoSYPS may in the future represent a very effective alternative. This newly developed tool is considered as a versatile, cost-effective and time-efficient platform assessing the GMO presence in analytical samples. In addition, it functions in routine analysis for enforcement purposes in a commonly applied 96-well plate qPCR format.

In the future, the research of the molecular platform of the WIV-ISP will focus on the development of more discriminative SYBR®Green qPCR screening methods to cover the broad range of GMO and UGM and thus to improve the resolution of the system. Particular importance will be given to their use in a modular approach associated with a decision tree cascade. Moreover, our strategy aiming at developing harmonised SYBR®Green qPCR screening methods incorporated in the Combinatory SYBR®Green qPCR Screening (CoSYPS) system has a potential to be applied in other scientific fields than GMO detection. The application of this strategy for food borne pathogenic bacteria is now under development in our team.

## **8. Glossary**

394 Polymerase Chain Reaction

In the near future, the number and the diversity of GM crops will continue to increase, as well as the requests for authorisation for their import for food and feed in the EU. Beside the notifications of GM events produced by multinational biotech companies, many GM events will be developed by universities, national research centres and small private companies. Thus, the chance for accidental occurrence of unapproved GMO in the EU food and feed chain trough importation will be higher. As the EU's general policy supports strong commitment to consumer protection and freedom of choice, and therefore mandatory product labelling, the development of sensitive, reliable but also cost-effective and flexible strategies for the detection of GMO in products through establishment of molecular

The GMO detection platform developed at WIV-ISP consists of a pre-PCR step namely DNA extraction and three consecutive qPCR phases. In this view, the choice of efficient methods to extract good quality DNA, in particular for processed food and feed, is a critical factor. A pre-PCR evaluation of the extracted gDNA is necessary as well as setting criteria for the purity and integrity of the DNA. Furthermore, the presence of PCR inhibitors is a major obstacle for efficient amplification in qPCR. This step may even become more important as the number of GM plant taxa becomes larger. Developing simple standard methods for genomic DNA extraction minimizing inhibition will therefore be the key for providing

Due to the broad range of GMO that my occur in the EU food and feed chains, the use of screening strategies only based on the 35S promoter of the Cauliflower Mosaic Virus (p35S) and the nopaline synthase terminator of *Agrobacterium tumefaciens* (tNOS) followed by the analysis of the sample with event-specific EU validated methods by the enforcement laboratories will become insufficient. As a consequence, new methods focusing on an

At the present time several high-tech strategies like multiplex PCR and consecutive detection and identification of the amplification products using micro-arrays (Chaouachi et al., 2008, Morisset et al., 2008, Hamels et al., 2009) or PCR combined with capillary electrophoresis (Nadal et al., 2009) have been proposed to deal with this discriminative problem and the broad diversity of GMO. However, at the present time, these technologies require additional costly equipment and investments in technical support. Furthermore, they need technological optimalisation as they show a high background at low target level.

Contrary to the above-mentioned technically complex strategies, our approach based on numerous singleplex qPCR-based methods developed to function under the same reaction conditions combined with the informatics decision support tool CoSYPS may in the future represent a very effective alternative. This newly developed tool is considered as a versatile, cost-effective and time-efficient platform assessing the GMO presence in analytical samples. In addition, it functions in routine analysis for enforcement purposes in a commonly applied

In the future, the research of the molecular platform of the WIV-ISP will focus on the development of more discriminative SYBR®Green qPCR screening methods to cover the

These difficulties make them less suitable for routine or enforcement purposes.

**7. Conclusion**

platforms will become more and more crucial.

concordant results when using qPCR techniques.

intensive screening analysis need to be developed.

96-well plate qPCR format.

#### **Amplification Efficiency**

The amplification efficiency is the rate of amplification that leads to a theoretical slope of – 3,32 with an efficiency of 100% in each cycle. The efficiency of the reaction can be calculated by the following equation:

$$\text{Efficiency} = 10^{\left(\frac{-1}{\text{slope}}\right)} - 1 \tag{1}$$

### **Applicability**

Applicability is the description of analytes, matrices and concentrations to which the method can be applied.

#### **Certified Reference Material (CRM)**

A Certified Reference Material is a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability.

#### **Correlation Coefficient (R2)**

The R2 coefficient is the correlation coefficient of a (calibration) curve obtained by linear regression analysis.

#### **Dynamic Range**

The dynamic range is the range of concentrations over which the method performs in a linear manner with an acceptable level of trueness and precision.

#### **European Food Safety Authority (EFSA)**

EFSA is an agency of the EU that provides independent scientific advice and communication on all matters concerning food and feed safety.

#### **European Network of GMO Laboratories (ENGL)**

The European Network of GMO Laboratories is a platform of EU experts that plays an eminent role in the development, harmonisation and standardisation of means and methods for sampling, detection, identification and quantification of Genetically Modified Organisms (GMO) or derived products in a wide variety of matrices, covering seeds, grains, food, feed and environmental samples. The network was inaugurated in Brussels on December

Development of a Molecular Platform

**Reference material (RM)**

in a measurement process.

**Robustness**

**Specificity**

**Threshold cycle (Ct)**

usually expressed in terms of bias.

**9. Acknowledgement**

Science Policy.

interest.

**Trueness**

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 397

results are obtained with the same method, on identical test items, in the same laboratory,

The relative reproducibility standard deviation is the relative standard deviation of test results obtained under reproducibility conditions. Reproducibility conditions are conditions where the test results are obtained with the same method, on identical test items, in different laboratories, with different operators, using different equipment. Reproducibility standard

A Reference Material is a material that is sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use

The robustness of a method is a measure of its capacity to remain unaffected by small, but

Specificity is a property of a method to respond exclusively to the characteristic or analyte of

The threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The Ct value assigned to a particular well thus reflects the point during the reaction

The trueness is defined as the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. The measure of trueness is

The authors like to acknowledge Lievens Antoon for his critical review of the manuscript and De Keersmaecker Sigrid for her help with figures and layout. This work of the GMOlab was financially supported by four projects: the British Food Standard Agency (FSA, contract G03032) and the German Federal Office of Consumer Protection and Food Safety (BVL) through the Project GMOseek, under the European ERA-NET consortium SAFEFOODERA; the GMODETEC project (RT-06/6) of the Belgian Federal Ministry ''Health, Food Chain Safety and Environment'; the European Commission through the Integrated Project Co-Extra (Contract No. 007158), under the 6th Framework Program and by the Belgian Science Policy projects SPSD I & II of the Belgian Federal Ministry of

deliberate deviations from the experimental conditions described in the procedure.

at which a sufficient number of amplicons has been accumulated.

by the same operator, using the same equipment within short intervals of time.

**Precision – Relative Reproducibility Standard Deviation (RSDR%)**

deviation describes the inter-laboratory variation.

4th 2002 and it currently consists of more than 100 national enforcement laboratories, representing all 27 EU Member States plus Norway and Switzerland. Its plenary meetings are open to particular observers, such as to representatives from Acceding and Candidate Countries.

## **European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF)**

The core task of the EU-RL GMFF is the scientific assessment and validation of detection methods for GM Food and Feed as part of the EU authorisation procedure. The Joint Research Centre (JRC) of the European Commission and, more precisely, the Molecular Biology and Genomics Unit of the Institute for Health and Consumer Protection (IHCP), has been given the mandate for the operation of the EU-RL GMFF. Activities are carried out in close collaboration with European Network of GMO Laboratories (ENGL).

## **Genetically Modified (GM) event**

A GM event refers to the unique DNA recombination event that took place in one plant cell, which was then used to generate entire transgenic plants

## **Genetically Modified Organism (GMO)**

A Genetically Modified Organism is officially defined in the EU legislation as "organisms, not from human origin, in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination"

#### **Limit of Detection (LOD)**

The limit of detection is the lowest amount or concentration of analyte in a sample, which can be reliably detected but not necessarily quantified, as demonstrated by single-laboratory validation.

#### **Limit of Quantification (LOQ)**

The limit of quantification is the lowest amount or concentration of analyte in a sample that can be reliably quantified with an acceptable level of precision and accuracy.

### **Melting temperature (Tm)**

The melting temperature is the temperature at which 50% of the DNA is single stranded.

#### **National Reference Laboratory (NRL)**

A National Reference Laboratory on GMO operates in the frame of Commission Regulation EC/1829/2003 on GM Food and Feed and Commission regulation EC/1830/2003 on labelling and traceability of GMO. It assists the EU-RL and the NRL from the different member states are gathered in the ENGL.

#### **Practicability**

Practicability is the ease of operations, the feasibility and efficiency of implementation, the associated unitary costs (e.g. cost/sample) of the method.

#### **Precision - Relative Repeatability Standard Deviation (RSDr%)**

The relative repeatability standard deviation is the relative standard deviation of test results obtained under repeatability conditions. Repeatability conditions are conditions where test results are obtained with the same method, on identical test items, in the same laboratory, by the same operator, using the same equipment within short intervals of time.

## **Precision – Relative Reproducibility Standard Deviation (RSDR%)**

The relative reproducibility standard deviation is the relative standard deviation of test results obtained under reproducibility conditions. Reproducibility conditions are conditions where the test results are obtained with the same method, on identical test items, in different laboratories, with different operators, using different equipment. Reproducibility standard deviation describes the inter-laboratory variation.

#### **Reference material (RM)**

A Reference Material is a material that is sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process.

#### **Robustness**

396 Polymerase Chain Reaction

4th 2002 and it currently consists of more than 100 national enforcement laboratories, representing all 27 EU Member States plus Norway and Switzerland. Its plenary meetings are open to particular observers, such as to representatives from Acceding and Candidate

The core task of the EU-RL GMFF is the scientific assessment and validation of detection methods for GM Food and Feed as part of the EU authorisation procedure. The Joint Research Centre (JRC) of the European Commission and, more precisely, the Molecular Biology and Genomics Unit of the Institute for Health and Consumer Protection (IHCP), has been given the mandate for the operation of the EU-RL GMFF. Activities are carried out in

A GM event refers to the unique DNA recombination event that took place in one plant cell,

A Genetically Modified Organism is officially defined in the EU legislation as "organisms, not from human origin, in which the genetic material (DNA) has been altered in a way that

The limit of detection is the lowest amount or concentration of analyte in a sample, which can be reliably detected but not necessarily quantified, as demonstrated by single-laboratory

The limit of quantification is the lowest amount or concentration of analyte in a sample that

The melting temperature is the temperature at which 50% of the DNA is single stranded.

A National Reference Laboratory on GMO operates in the frame of Commission Regulation EC/1829/2003 on GM Food and Feed and Commission regulation EC/1830/2003 on labelling and traceability of GMO. It assists the EU-RL and the NRL from the different

Practicability is the ease of operations, the feasibility and efficiency of implementation, the

The relative repeatability standard deviation is the relative standard deviation of test results obtained under repeatability conditions. Repeatability conditions are conditions where test

can be reliably quantified with an acceptable level of precision and accuracy.

**European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF)**

close collaboration with European Network of GMO Laboratories (ENGL).

which was then used to generate entire transgenic plants

does not occur naturally by mating and/or natural recombination"

Countries.

**Genetically Modified (GM) event**

**Limit of Detection (LOD)**

**Limit of Quantification (LOQ)**

**National Reference Laboratory (NRL)**

member states are gathered in the ENGL.

associated unitary costs (e.g. cost/sample) of the method.

**Precision - Relative Repeatability Standard Deviation (RSDr%)**

**Melting temperature (Tm)**

validation.

**Practicability**

**Genetically Modified Organism (GMO)**

The robustness of a method is a measure of its capacity to remain unaffected by small, but deliberate deviations from the experimental conditions described in the procedure.

#### **Specificity**

Specificity is a property of a method to respond exclusively to the characteristic or analyte of interest.

### **Threshold cycle (Ct)**

The threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The Ct value assigned to a particular well thus reflects the point during the reaction at which a sufficient number of amplicons has been accumulated.

#### **Trueness**

The trueness is defined as the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. The measure of trueness is usually expressed in terms of bias.

## **9. Acknowledgement**

The authors like to acknowledge Lievens Antoon for his critical review of the manuscript and De Keersmaecker Sigrid for her help with figures and layout. This work of the GMOlab was financially supported by four projects: the British Food Standard Agency (FSA, contract G03032) and the German Federal Office of Consumer Protection and Food Safety (BVL) through the Project GMOseek, under the European ERA-NET consortium SAFEFOODERA; the GMODETEC project (RT-06/6) of the Belgian Federal Ministry ''Health, Food Chain Safety and Environment'; the European Commission through the Integrated Project Co-Extra (Contract No. 007158), under the 6th Framework Program and by the Belgian Science Policy projects SPSD I & II of the Belgian Federal Ministry of Science Policy.

Development of a Molecular Platform

16

102, p.14-25

p.9-15

*Diseases*, Vol. 46, No. 3, pp. 189-195, 0732

*alimentarius commission – WHO*. Rome.

*the European Union*, L 191, p.1-141

*European Union*, L 268, pp.1-23

*Journal of the European Union*, L 268, pp. 24-28

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Cloud, J.L., Hymas, W.C., Turlak, A., Croft, A., Reischl, U., Daly, J.A. & Carroll, K.C. (2003).

Codex Alimentarius Commission - Joint FAO/WHO Food Standards Programme - Codex

Commission Recommendation (EC) No 787/2004 of 4 October 2004 on technical guidance

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Commission Regulation (EC) No 619/2011 of 24 June 2011 laying down the methods of

Commission Regulation (EC) No 882/2004 of the European Parliament and of the council of

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Commission Regulation (EC) No 1981/2006 of 22 December 2006 on detailed rules for the

Description of a multiplex *Bordetella pertussis* and *Bordetella parapertussis* LightCycler PCR assay with inhibition control. *Diagnostic Microbiological infectious*

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**19**

*1,2,3,4,5,7Iran 6Turkey*

**Overview of Real-Time PCR Principles**

*1Laboratory of Genetics, Legal Medicine Organization of Tabriz, Tabriz,*

*5Faculty of Medicine, Shahid Behesti University of Medical Sciences, Tehran,*

*7Department of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz*