PCR-based GM Diagnostics: Strategies for Detection of Genetically Modified Events
Authors: Sushmita, Dr. Monika Singh (Co-author)

Genetically modified (GM) crops are being commercially cultivated worldwide, possessing major agriculturally important traits like abiotic and biotic stress resistance, better yield and enhanced nutritional profile etc. in order to ensure global food security. With increase in commercialization of such crops, presence of genetically modified organisms (GMOs) in crops and products can be regulated in a systematic manner by employing GM diagnostics.

What are GMOs?

GMOs or genetically modified organisms are any biological entities whose genetic material has been altered for the trait of interest through the use of genetic engineering.

Constituents of a Gene construct:

  • Promoter acting as the regulatory switch to drive the gene expression
  • Transgene encoding the specific trait of interest to be incorporated in the host
  • Terminator as the stop signal
  • Marker genes for differential expression of the transformed entity in comparison to the non-transformed ones

    Major components of a transgenic construct

    Levels of GM Diagnostics

  • Detection : To check the GM status of a sample. If the result is GM positive, then further processing is done through subsequent steps.
  • Identification : If the sample is found to be GM then the identity of the GM crop is to be established.
  • Quantification : In order to comply with the regulatory requirements, the amount of GM content in a sample is to be estimated. Thus, the final level is to quantify the GM content in the identified crop.

GM Detection Strategies employed for Routine Testing:

Two methods are broadly employed for GM detection:

  1. - Protein-based methods
  2. - DNA-based methods
Protein-based GM detection methods are based on antigen-antibody interaction through immunoassays like ELISA (Enzyme Linked Immunosorbent Assay), lateral flow strips and western blot. These are highly specific for detection of novel protein which is expressed as a result of genetic transformation. The limitation of these immunoassays vests with the fact that in case of processed food the protein may get degraded and thus cannot be expressed.

DNA-based methods are relatively more stable, robust and sensitive than protein-based assays and have the following variants:
  1. - PCR-based methods
  2. - Real-Time PCR based methods

PCR-based GM Detection Methods

PCR-based GM Detection methods involve amplification of specific region of the integrated transgenic construct and based on the specificity required, can be categorised as under:
  • Screening methods : Involve the detection of presence or absence of GMO in test samples. For such methods, commonly used genetic elements of transgenic construct are employed as the target to be detected. This constitutes the preliminary step in GM detection as it would help to screen out the GM events from a given test sample. The screening elements can be exemplified as: p35S ( Cauliflower Mosaic Virus 35S promoter) and Tnos (nopaline synthase terminator obtained from Agrobacterium tumefaciens). There are several other commonly used targets as well like Figwort Mosaic Virus promoter (pFMV), A. tumefaciens nopaline synthase promoter (pNos).
  • Gene-specific methods: Target the transgenes specific to a particular trait of an event and are, therefore, more specific with respect to screening methods. Once the preliminary step of screening is over, we can find out the gene present in the sample, which is specific to a particular event. Some of these genes in major GM events are: cry1Ac gene in Bollgard I event of cotton for insect resistance; cry2Ab in Bollgard II event of cotton in addition to cry 1Ac for Insect resistance, cp4-epsps in Roundup Ready event of soybean.
  • Construct-specific methods : The junction sequences between two adjoining DNA segments constitutes the construct-specific method of GM detection. The junction may be the point spanning between promoter and transgene. Some of the examples of construct-specific methods are:ctp2-cp4epsps, p35S-cry1Ac and cry1Ac-cry1Ab.
  • Event-specific methods: This method is the most specific method for GM detection as it targets the site of insertion in the genome locus even if the same construct is inserted. Based on the site of integration, specific GM events could easily be identified.
The increasing number of area under GM crops and trait diversification require an efficient monitoring method and thus, the GMO testing laboratories require to have the cost-efficient technologies with validated protocols for GM diagnostics.

About Author / Additional Info:
We both are working as Scientist at ICAR-National Bureau of Plant Genetic Resources, New Delhi