Detection of genetically modified plants By: Ehsan Zayerzadeh Standard Research Institute

Transgenic plants • Characterized by the insertion of a new gene or sets of genes into their genome • The new genes translate and new protein expressed • This gives the plant new characteristic

Some Transgenic plants • Bt cotton – Cotton plants resistant to lepidopteran insects • Round-up ready soybean – Soybean resistant to glyphosate • Golden rice – Rice grains with beta-carotene

Introduction *Transgenic crops offer significant production Advantages: It reduces the cost of production It escapes environmental pollution due to the indiscriminate use of pesticides and herbicides. *The first and most important reason of regulation Human safety protect the environment

How transgenic plants are made? Identification of useful gene The cloning of the gene into a suitable plasmid vector, Delivery of the vector into plant cell. A gene construct consists typically of three elements: The promoter The terminator The transgene encodes a specifically selected trait

Introduction

Introduction For every country expected to use genetically engineered crops or food products resulted from them, detection capacity should be readily available.

Introduction ISO 21569:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Qualitative nucleic acid based methods (9617) ISO 21570:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Quantitative nucleic acid based methods ISO 24276:2006 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- General requirements and definitions (9613) ISO 21571:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Nucleic acid extraction (10763) ISO 21568: Foodstuffs- Methods of analysis for the detection of genetically modified organisms and derived products-Sampling

Introduction Sampling Sample preparation Analytical method * Sampling expert Sample Size (systematic sampling ) specialized sampling equipment correct labels, forms Storage Transportation

Detection strategies The basis for every type of GMO detection technology is to exploit the difference between the unmodified variety and the transgenic plant Detection of a GMO can be done by detecting a molecule (DNA, RNA or protein) that is specifically associated with or derived from the genetic modification of interest

Detection strategies The majority of the methods developed for detection of GMOs and GMO-derivatives focus on detecting DNA, while only a few methods have been developed for detecting proteins or RNA For detection, the availability of validated methods, availability of reference material, and the homogeneity of the sample are crucial parameters

Detection strategies DNA-based approaches Protein-based approaches

DNA-based approaches PCR method These method is only to detect the presence or absence of a foreign gene in a sample Real-time PCR Real-time PCR measures the amount of molecules produced during each stage of the PCR rather than just at the end Micro-array – Automated rapid screening – Based on nucleic acid hybridisation – Specific probes are attached to the chip

DNA extraction and purification CTAB method DNeasey® (Qiagen, Hilden, Germany) protocol i-Pure™ GMO Extraction Kit (both from Bio-Rad Laboratories Inc.) G-spin™ for plant, Genomic DNA extraction kit (iNtRON Biotechnology( The concentration of the extracted DNA can be determined spectrophotometricaly (UV 260/280) or fluorometricaly.

Protein-based approaches Western blot. Western blot is a highly specific method that provides qualitative results suitable for determining whether a sample contains the target protein below or above a predetermined threshold level. ELISA. ELISA involves testing for the presence of specific proteins by exploiting the specificity of binding between expressed antigen and target antibody Lateral flow strip or strip test. Strip test is considered as a version of ELISA

DNA-based approaches Merits The major advantage of PCR tests is sensitivity, ie, detection of GMOs at very low levels. • Work with most product types - both processed and unprocessed products • DNA molecule is relatively stable during processing A minimum amount of sample material containing intact DNA

DNA-based approaches Demerits Extremely sensitive, very low levels of inadvertent contamination can result in false positives Sample analysis requires longer time(approximately one day). Compared with protein-based methods, equipment and operation are costly, and highly trained personnel are required

Protein-based approaches Merits Protein strip tests are simple, fast, cheap and reliable, making them a complementary tool to the PCR GMO detection methods • Low per sample cost once reagents are developed. • Less sensitive than PCR, therefore, less susceptible than PCR to false positives caused by minor levels of contamination. • Strip tests can be used in the field

Protein-based approaches Demerits • Protein denaturation as a consequence of processing detection methods • High up-front costs for assay development and generation of antibodies and protein standards. • Not appropriate for processed products Not very sensitive.

Conclusion Strip test and PCR method are the best methods for detection of GM plants The quantitative PCR approaches and ELISA are the best methods for quantifying GM plants

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