DNABased Detection

DNA-based detection of transgenic plants targets the novel DNA sequences introduced into the crop genome. These methods show the absence or presence of GM plant material in a sample and can also measure the relative quantity (percentage) in a tested sample.

  1. 7.1 Analytical strategy and targeted sequences. Detection of genetically modified plants in food, feed and seed samples is generally conducted by consecutive PCR tests targeting the genetic elements (element-specific) and constructs (construct-specific). For event-specific identification and quantification of GM plants the 5' or 3' junction regions around the integration sites are targeted. A plant taxon-specific reference gene is targeted for relative quantification of the GM content. Element- and construct-specific methods are applicable mainly for screening purposes, event-specific methods are required for identification and quantification
  2. 7.1 Analytical strategy and targeted sequences. Detection of genetically modified plants in food, feed and seed samples is generally conducted by consecutive PCR tests targeting the genetic elements (element-specific) and constructs (construct-specific). For event-specific identification and quantification of GM plants the 5' or 3' junction regions around the integration sites are targeted. A plant taxon-specific reference gene is targeted for relative quantification of the GM content. Element- and construct-specific methods are applicable mainly for screening purposes, event-specific methods are required for identification and quantification
  3. 2.1.1 Polymerase Chain Reaction

DNA-based testing for GM plants is commonly performed using PCR, amplifying specifically a short segment of the targeted DNA (Fig. 7.1). The design of specific primers depends on a knowledge of the precise and comprehensive DNA sequence information of the actually integrated DNA. If the method is to detect specifically a certain transformation event, information about the inserted DNA sequence and the 3' and 5' flanking plant genome sequences is required. For element-specific PCR-based screening and construct-specific detection the DNA sequences of the inserted elements and gene constructs are targeted, respectively.

PCR-based detection and particularly the quantitative measurement of the GM content in a sample actually involves the use of two PCR systems, one for determination of the inserted GM-derived DNA sequence and another system specific for an endogenous, plant-taxon specific reference gene sequence (Fig. 7.1). The latter is also thought to serve as a control for the quality and quantity of the extracted DNA.

7.2.1.2 Conventional Qualitative PCR

Conventional PCR methods are mainly used for qualitative testing to obtain yes/no answers concerning the presence of GM plant material. PCR products are analysed by agarose or polyacrylamide gel electrophoresis (Sambrook and Russel 2001) and visualised using UV fluorescence with ethidium bromide as fluorophor or by other means. It may be necessary to confirm GM-positive test results by further analyses, either by restriction analyses, Southern hybridisation or DNA sequencing (ISO 2005a).

Before the PCR method is applied the primer combination has to be optimised and validated for their performance requirements. The important performance criteria for qualitative PCR methods are the sensitivity in detecting the transgenic DNA sequences and the specificity for the targeted DNA segment. At optimal reaction conditions a limit of detection (LOD) of 1-10 copies of the target sequence can be achieved in less than 40 PCR cycles (Hiibner et al. 2001). Practically the LOD of the PCR method should allow that the presence of the target sequence is detected in at least 95% of the time, with < 5% false negative results (ENGL 2008). The length of the amplified product influences the PCR performance and should therefore selected in a way that it matches to the size range of DNA fragments which can be extracted from the sample matrix. For raw materials like seeds or leaves containing less fragmented DNA a broader range of PCR product size up to maximally 250 bp is applicable, whereas for processed food or feed with higher DNA fragmentation the PCR product should be ideally 80-150 bp. The specificity of the method should be tested theoretically by sequence similarity search with the primer sequences against nucleic acid sequence databases (e.g. Blast search in EMBL, GenBank, etc.) and empirically by testing the GM target event(s), very similar nontarget GM events and different non-GM plants in order to confirm that the primers can discriminate between the target and closely related non-target sequences. For the reference gene-specific PCR methods different varieties should be tested to demonstrate that the target sequence is conserved between different plant lines (Hernandez et al. 2004, 2005; Broothaerts et al. 2008).

7.2.1.3 Quantitative Real-Time PCR

The most preferred technique to quantify GM material in a sample is real-time PCR. It allows the detection and measurement of increasing fluorescence proportional to the amount of amplification products generated during the PCR process. Of the various chemistries TaqMan fluorogenic probes (Holland et al. 1991) are most commonly applied in real-time PCR-based detection and quantification of GM plant materials. Real-time PCR is mainly used for quantification purposes, but it is increasingly utilised also for qualitative testing to screen or to identify the GM event (Zeitler et al. 2002; Rho et al. 2004; Reiting et al. 2007; Waiblinger et al. 2007).

The limit of quantitation (LOQ) of a real-time PCR method depends on the optimisation of the PCR detection method and on the accepted standard deviation of the measurement. The LOQ is experimentally determined during method validation and should reach 30-50 target molecules, which is close to the theoretical prediction (Hubner et al. 2001). As shown in Table 7.1, the LOD/LOQ values depend primarily on the characteristic plant genome size (C value) and range from 0.004%/0.02% for papaya to 0.16%/0.7% for wheat. The obvious effect here is that PCR is inhibited when the amount of input DNA is exceeding approx. 8 ng/p.l of reaction volume. For example for maize, according to its genome size a 200-ng DNA sample contains approximately 39 000 genome copies and thus a given sample with a GM plant content of 0.1% corresponds to 39 copies for a single-copy

Table 7.1 Plant genome size and theoretical LOD/LOQ in real-time PCR assays

Common

Scientific name

Nuclear DNA

Genome

LODc

LOQ

name

contenta

copies

Mbp/1C

pg/2Cb

(in 200 ng)

(%)

(%)

Alfalfa/

Medicago sativa (2n=4X)

1.510

3.09

64 768

0.02

0.06

lucerne

Barley

Hordeum vulgare

4.873

9.97

20 070

0.05

0.2

Cotton

Gossypium hirsutum

2.246

4.59

43 544

0.02

0.1

Maize

Zea mays

2.504

5.12

39 058

0.03

0.1

Oilseed rape

Brassica napus

1.182

2.42

82 741

0.01

0.05

Papaya

Carica papaya

0.372

0.76

262 903

0.004

0.02

Pea

Opisum sativum

4.172

8.53

23 442

0.04

0.2

Peanut

Arachis hypogaea(2n=4X)

2.813

5.75

34 767

0.03

0.1

Potato

Solanum tuberosum (2n=4X)

1.730

3.54

56 548

0.02

0.07

Soybean

Glycin max

1.115

2.28

87 713

0.01

0.05

Sugarbeet

Beta vulgaris ssp. Saccharifera

0.758

1.55

129 024

0.01

0.03

Sunflower

Helianthus annuus

3.030

6.20

32 277

0.03

0.1

Tobacco

Nicotiana tabacum (2n=4X)

4.434

9.07

22 059

0.05

0.2

Tomato

Lycopersicon esculentum

0.954

1.95

102 569

0.01

0.04

Rice

Oryza sativa

0.441

0.90

221 769

0.005

0.02

Wheat

Triticum aestivum (2n=6X)

15.966

32.65

6 126

0.16

0.7

d aNuclear DNA content values were taken from Arumuganathan and Earle (1991) b1 picogram (pg) = 978 x 106 base pairs (Dolezel et al. 2003)

cRelative limit of detection (LOD) based on an LOD (CI=95%) of 8-12 copies of the GM target sequence (Burns and Valdivia 2008)

drelative limition of quantification (LOQ) based on an LOQ of 40 copies for the GM target sequence (Hubner et al. 2001)

aNuclear DNA content values were taken from Arumuganathan and Earle (1991) b1 picogram (pg) = 978 x 106 base pairs (Dolezel et al. 2003)

cRelative limit of detection (LOD) based on an LOD (CI=95%) of 8-12 copies of the GM target sequence (Burns and Valdivia 2008)

drelative limition of quantification (LOQ) based on an LOQ of 40 copies for the GM target sequence (Hubner et al. 2001)

d transgene. A quantitative real-time PCR assay should be carefully optimised for the specific LOD/LOQ needed for GM content detection and quantification. The precision of the quantitative real-time PCR methods is commonly expressed as relative standard deviation (RSD) which can vary over 10-30% with respect to intra-laboratory repeatability and over 15-50% for inter-laboratory reproducibility, depending on the range of target copies analysed.

7.2.1.4 Alternative DNA-Based Techniques

To solve the challenge that the increasing number of GM plant events is covered by appropriate analytical methodologies it is expected that multi-target analyses are necessary. The DNA microarray technology could be an option to parallelise the multi-analyte detection of several PCR products in a single run. Arrays that have been developed consist of various oligonucleotide probes that are immobilised on a glass support and used for screening of genetic elements, for constructs and events including detection of plant taxon-specific reference genes (Hamels et al. 2007; Xu et al. 2007; Leimanis et al. 2008). However, this approach is based on the use of multiplex PCR before hybridisation of the PCR products to the microarray and, as has been shown elsewhere, PCR is limited in its multiplexing capacity within one reaction due to the reduced sensitivities of the individual PCR systems. Therefore, alternative amplification methods are currently investigated for their potential use for GMO detection in the future, particularly to cover the increasing number of GM host plants and diversity in genetic elements and constructs. Several alternatives are being tested for improvements in GMO detection, e.g. loop-mediated isothermal amplification (LAMP; Fukuta et al. 2004), ligation-depended probe amplification (LPA; Moreano et al. 2006), SNPlex technology (Chaouachi et al. 2008), padlock probe ligation in combination with microarray detection (Prins et al. 2008) and nucleic acid sequence based amplification using transcription techniques (NASBA) in combination with microarray detection (Morisset et al. 2008). In addition, to circumvent the limitations concerning the availability or reference materials (e.g. for unauthorised GM events), the use of multiple displacement amplification (MDA) for whole-genome amplification has been described to generate reference material for GMO detection (Roth et al. 2008).

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